Modified nuclear glucocorticoid receptor, fusion protein, and DNA fragments coding for said receptor and said fusion protein

ABSTRACT

A DNA fragment coding for a modified nuclear glucocorticoid receptor, particularly one mutated in the region coding for the ligand binding domain, so that receptor activity is more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligand, is disclosed. A recombination system inducible in mammals by means of a fusion protein produced between a recombinase and the binding domain of the ligand derived from the modified glucocorticoid receptor of which the activity is more strongly inducible by synthetic glucocorticoids than by natural glucocorticoids, is also disclosed.

FIELD OF THE INVENTION

The present invention relates to a DNA fragment coding for a modifiednuclear glucocorticoid receptor (GR), and a DNA fragment coding for aligand binding domain (LBD) of said receptor, as well as a DNA fragmentcoding for a fusion protein comprising said receptor of said ligandbinding domain of said receptor fused to a protein whose activity isinduced in the presence of glucocorticoid ligands.

The present invention also relates to a modified nuclear glucocorticoidreceptor, in particular the human receptor and its modified ligandbinding domain, as well as a fusion protein comprising said receptor orsaid ligand binding domain.

The present invention also relates to a vector for conditionallyexpressing a protein, in particular a foreign protein, in human oranimal host cells, in particular mammalian cells. The present inventionalso relates to a method of expressing a protein in said cells.Moreover, the present invention relates to a method for the conditionalexcision, insertion, inversion or translocation of a DNA fragment inhuman or animal, in particular mammalian, host cells.

The present invention relates in addition, to a vector for transferringa DNA fragment into said human or animal, in particular mammalian, hostcells and to its use as a medicament, as a tool for analyzing andstudying the function of a gene as well as to a method of treating cellsex vivo or in vitro.

Finally, the present invention relates to human or animal cellstransfected with an expression and/or transfer vector according to theinvention as well as to transgenic animals derived therefrom.

BACKGROUND OF THE INVENTION

The glucocorticoid receptor (GR) is a protein for regulatingtranscription of genes which mediate the action of glucocorticoids intarget cells. The GR and other members of the super family of nuclearreceptors (ligand-activated transcription factors) exhibit a commonmodular structure. The variable N-terminal region contains theconstitutive transactivation activity AF-1. The core DNA binding domain(DBD) is highly conserved between various species and allows the bindingof the receptor to its related DNA response element (GRE in the case ofGR). The ligand binding domain (LBD), located in the C-terminal regionof GR, not only binds the ligand, but also comprises multiple distinctactivities: a surface for interaction with hsp90 and a distinct surfacefor homodimerization, a nuclear localization signal and aligand-dependent transactivation function AF-2 (for review articles andreferences, see (1-5)). The LBD domain is highly conserved betweenvarious species. A transactivation function AF-2 has been identified inthe C-terminal part of various LBDs (6-10). The integrity of the corepart of the region containing AF-2 is required for the ligand-dependenttranscription activation by the corresponding nuclear receptors, and forthe interaction between this region and related transcriptionalintermediate factors (TIF, also called coactivators or mediators)(11-19). A general model has been proposed (19) in which the binding ofthe ligand induces a conformational change in the ligand binding domainby calling into play in particular the C-terminal region harboring thecore part of AF-2, thus generating a surface for an effectiveinteraction with the TIFs. The LBDs of the nuclear receptors containconserved regions possessing a canonic structure In the form of a helix.These structural data have made it possible to carry out a commonalignment or the LBDs of all nuclear receptors. The H12 helix containsthe transactivation function AF-2 (19). The m no acids of helices H11and H12, in particular of the glucocorticoid receptors from variousspecies such as humans, rats, mice and xenopus, are conserved.

Several mutations in the LBD of GR have been previously described. Twomajor types of mutation can be distinguished:

the first type consists in mutations which positively or negativelyaffect the affinity for binding to the ligand. For example, thereplacements of the cysteine residue 656 by glycine in rat GR (20) andof the methionine residue 565 by arginine or of the alanine residue 573by glutamine in the human GR (hGR) (21) increase the affinity forbinding to the ligand and lead to a shift in the dose-response curve forthe transactivation in the direction of the lowest ligandconcentrations. These mutants are consequently designated “super GR”.Likewise, mutants have been reported which have a lower affinity forbinding to the ligand (21-24). LBD mutations which shift the liganddose-response curve in the direction of the highest ligandconcentrations result from an altered hormone binding affinity (25-26);

the second group of LBD mutants comprises those which affect thetranscriptional activation function (AF-2) without altering the bindingto the ligand. As examples, there may be mentioned mutations in theregion containing AF-2 which are linked to a loss or a decrease in thetransactivation potential, but do not affect the affinity for binding tothe ligand (6-10).

The fusion of the ligand binding domain (LBD) of nuclear receptors toheterologous proteins has made it possible to control their activity innumerous cases.

The activities of Myc, c-Abl, Src, erbB1, Raf, E1A Cre and FLP may bemodulated by producing fusion proteins with the LBD of nuclear receptors(27-29). However, the ligands for these receptors are present innumerous biological systems and are thus capable of inducing a basalactivity level. To avoid such problems, a mutated LBD of the estrogenreceptor (ER) has been fused to the c-Myc protein (30), or to the Creand FLP recombinases (28 and 29).

The recombinases of the family of λ integrases catalyze the excision,insertion, inversion or translocation of DNA fragments at the level ofspecific sites of recognition of said recombinases (31-36). Therecombinases are active in animal cells (35).

The Cre recombinase, an integrase of 38 KDa from bacteriophage P1,catalyzes recombination between two DNA sequences of 34 base pairscalled loxP in the absence of cofactors (32, FIG. 1).

The position on one or more DNA molecules and the orientation of loxPsites relative to each other determine the type of function of therecombinase, excision, insertion, inversion or translocation, inparticular the Cre recombinase (FIG. 1). Thus, the recombinase activityof Cre is an inversion when two LoxP sites are head-to-tail on the sameDNA fragment and an excision when the LoxP sites are a direct repeat onthe same DNA fragment. The recombinase activity is an insertion when aloxP site is present on a DNA fragment, it being possible for a DNAmolecule such as a plasmid containing a loxP site to be inserted at thelevel of said loxP site (FIG. 1). The Cre recombinase can also induce atranslocation between two chromosomes provided that a loxP site ispresent on each of them (37) (FIG. 1). More generally, the Crerecombinase is therefore capable of inducing recombination between oneor more different DNA molecules provided that they carry loxP sites.

Likewise, the FLP recombinase, a recombinase of 43 KDa fromSaccharomyces cerevisiae, is capable of the same type of action on DNAfragments containing recognition sites FRT (34).

By producing a fusion protein (chimera) between the Cre recombinase andthe C-terminal region of the human receptor for estrogens containing aValine at position 400, a molecule is obtained which is capable ofexcising, in the presence of the receptor ligand, estradiol, the DNAsequences located between two loxP sites, as well as one of the loxPsites, when the latter are a direct repeat, whereas in the absence ofligand, the excision does not take place (28).

The activity of the FLP recombinase can also be regulated by producingchimeras between the FLP and the binding domain of nuclear receptors. Byfusing the FLP with the LBD of the estrogen receptor or of theglucocorticoid receptor in the absence of ligand, the recombinaseactivity is very low, whereas it is rapidly induced by the respectiveligands for the LBDs (29).

However, the use of fusion proteins between recombinases and LBDs ofnuclear receptors to excise, in a controlled manner, DNA fragmentssituated between sites of recognition of said recombinases can pose aproblem, given that the ligand concentrations present in animals or incell culture media may be sufficient to induce, at least partially, therecombinase activity.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a recombination system inducible inanimals, in particular mammals, by virtue of the use of a fusion proteinproduced between a recombinase and the ligand binding domain derivedfrom the glucocorticoid receptor whose activity is inducible bysynthetic glucocorticoids but not by natural glucocorticoids inparticular at concentrations of less than 10⁻⁶ M, that is to sayphysiological concentrations.

To avoid induction of the recombinase activity of the chimeric proteinby the endogenous ligands, the present invention indeed proposes toprovide a mutation in the LBD of the glucocorticoid receptor allowing Itto be more strongly activated in the presence of syntheticglucocorticoid ligands than in the presence of natural glucocorticoidligands at physlologlcal concentrations.

The subject of the present invention is indeed a modified nuclearglucocorticoid receptor, in particular mutated in the region of theligand binding domain, so that the activity of said receptor is morestrongly inducible by a synthetic glucocorticoid ligand than by anatural glucocorticoid ligand.

According to the present invention, “(mutated) glucocorticoid receptormore strongly inducible by a synthetic glucocorticoid ligand than by anatural glucocorticoid ligand” is understood to mean that the activityof the mutated receptor according to the invention is induced morestrongly by synthetic glucocorticoid ligands than by naturalglucocorticoid ligands at a given concentration and that the mutatedglucocorticoid receptor according to the invention is a lot lesssensitive to the natural glucocorticoids than the wild-type receptor.

“Activity of said receptor” is understood to mean here thetranscriptional activity of said receptor and/or, where appropriate, theactivity of regulating the activity of a protein fused to said receptoror to said ligand binding domain, induced in the presence ofglucocorticoids. There may be mentioned more particularly the activityof a recombinase protein fused to said receptor or to said ligandbinding domain of said receptor.

“Natural glucocorticoids” are understood to mean here steroid hormonessecreted by the cortex of the adrenal glands such as cortisol,corticosterone or aldosterone. “Synthetic glucocorticoids” areunderstood to mean chemically synthesized agonists of said receptor forthe relevant activity. There may be mentioned, among the letter,dexamethasone, triamcimolone acetonide RU 486, RU 28362, bimedrazol,fluocinolone acetonide.

Some synthetic ligands will be agonists for inducing said particularactivity of the receptor and antagonists in relation to another activityof the receptor. Thus, the compound RU 486 induces the activity of arecombinase protein fused to said receptor or to said ligand bindingdomain of the receptor but does not induce the transcriptional activityof the receptor.

The synthetic glucocorticoid ligand may bind to the (nonmutated)wild-type nuclear receptor, but it is possible that said syntheticligand binds to the mutated receptor and not to the natural receptor. Itis also possible that if said synthetic ligand binds to the wild-typereceptor, the activity of the latter is nevertheless not induced bybinding to the synthetic ligand.

A mutation has in particular been identified in the LBD domain of thenuclear glucocorticoid receptors (isoleucine→threonine) situated betweenhelices H11 and H12 (19). This mutation causes a loss of activity forthe receptor in the presence of natural glucocorticoids. More precisely,this glucocorticoid receptor mutated between H11 and H12 has no or verylittle transcriptional activity in the presence of naturalglucocorticoids at their natural physiological concentrations, inparticular concentrations of less than 10⁻⁶ M. Its activity may,however, be induced by synthetic glucocorticoid ligands, in particulardexamethasone (dex).

The subject of the present invention is more particularly a mutatedhuman receptor called hGR (I747/T) characterized in that it has, assequence, the amino acid sequence of the human wild-type nuclearglucocorticoid receptor with an isoleucine→threonine mutation atposition 747, and still more particularly a modified human nuclearreceptor which has, as amino acid sequence, a sequence substantially asrepresented in the SEQ ID NO: 1.

The isoleucine 747 to threonine mutation in the C-terminal part of theligand binding domain (LBD) of the glucocorticoid receptor alters thecapacity of the natural ligands to induce the transactivation activitythereof. Natural glucocorticoids such as cortisol, aldosterone orcorticosterone are very weakly active or completely inactive with the GRmutant (I747/T), whereas synthetic glucocorticoids such as dexamethasone(Dex) efficiently stimulate the GR-mediated transactivation (I747/T).However, the corresponding ligand dose-response curve, for thetransactivation induced by Dex, is shifted toward the highestconcentrations, compared with that obtained with the wild-type (WT) GR.Neither the shift nor the inability of cortisol to efficiently activateGR (I747/T) are due to an altered affinity for binding to the ligand invitro.

Indeed, the mutation in the LBD of GR (I747/T) does not substantiallyalter the affinity for binding to the ligand in vitro. However, thismutant exhibits a substantial shift compared with the WT GR for theactivity curve toward the high ligand concentrations.

Moreover, so-called physiological concentrations of naturalglucocorticoid ligands which are sufficient to activate the WT GR causeonly a residual or zero activity in the mutant GR.

This mutant differs from the other GR LBD mutants previously described(22, 38) whose altered activation function is closely correlated with amodification in their properties for binding to the ligand in vitro.

The subject of the present invention is also a ligand binding domain ofthe nuclear glucocorticoid receptor according to the invention andparticularly the LBD domain [GR(I747/T)], characterized in that it hasan isoleucine→threonine mutation between helices H11 and H12. Moreparticularly, it has, as sequence, the ainmo acid sequence of the ligandbinding domain of the human glucocorticoid receptor substantially asrepresented in SID No. 1 from amino acid 532 to amino acid 777 with anisoleucine→threonine mutation at position 747.

The present invention also provides a DNA fragment encoding a modifiednuclear glucocorticoid receptor, in particular mutated in the region ofthe ligand binding domain (LBD) according to the invention, as well as aDNA fragment encoding a ligand binding domain (LBD) of the modifiedreceptor according to the invention.

The subject of the present invention is therefore more precisely a DNAfragment encoding a wild-type nuclear glucocorticoid receptor with anisoleucine to threonine mutation situated between helices H11 and H12 ofthe amino acid sequence of said receptor, and still more particularly aDNA fragment, characterized in that it has, as sequence, a codingsequence of the human nuclear glucocorticoid receptor whose amino acidsequence is substantially that represented in SEQ ID NO: 1.

In one embodiment, the subject of the present invention is a DNAfragment, characterized in that it has, as sequence, the cDNA sequenceof the human nuclear glucocorticoid receptor substantially asrepresented in SEQ ID NO: 1 comprising the ACC codon coding forthreonine at position 747 of the amino acid sequence of said receptor.

Likewise, the subject of the present invention is a DNA fragmentencoding the ligand binding domain (LBD) of the modified nuclearglucocorticoid receptor with an isoleucine→threonine mutation situatedbetween helices H11 and H12 and in particular at a positioncorresponding to position 747 of the amino acid sequence of the humannuclear glucocorticoid receptor.

More particularly, the present invention provides a DNA fragmentencoding the ligand binding domain of the modified human nuclearglucocorticoid receptor, characterized in that it has, as sequence, asequence coding for the amino acids of the ligand binding domain of thehuman nuclear glucocorticoid receptor whose amino acid sequence issubstantially as represented in SEQ ID NO: 1 from amino acid 532 toamino acid 777.

Still more particularly, the present invention provides a DNA fragment,characterized in that it has, as sequence, the cDNA sequence encodingthe ligand binding domain (LBD) of the human nuclear glucocorticoidreceptor substantially as represented in SEQ ID NO: 1 from codon 532 tocodon 777 with the ACC codon at position 747.

The subject of the present invention is, in addition, a vector systemfor conditionally expressing a protein, in particular a foreign protein,in host cells comprising an element for control of transcriptioninducible by a complex formed by a nuclear glucocorticoid receptor and aligand, characterized in that it comprises:

a first DNA fragment consisting of a DNA fragment coding for saidprotein under the control of elements ensuring its expression in saidhost cells, said elements ensuring its expression comprising a sequencefor control of transcription (RE) inducible by the receptor according tothe invention complexed with a synthetic glucocorticoid ligand, and

a second DNA fragment consisting of a functional DNA fragment coding forsaid receptor according to the invention or only part of said fragmentcomprising the region which recognizes the ligand (LBD) according to theinvention, and the DNA binding region (DBD) which attaches to saidsequence for control of transcription (RE),

it being possible for said first and second DNA fragments to be carriedby the same vector or two vectors separately.

Some cells have endogenous GR which may be activated by natural ligandsand synthetic ligands. If it is desired to activate a given gene at willin such cells, without it being possible for it to be activated byendogenous ligands or by endogenous receptors, it is necessary for thisgene to contain an RE different from GRE and to use a chimeric activatorcomprising the corresponding DBD and a mutated LBD according to theinvention. This gene can therefore be activated by synthetic ligands,but not by natural ligands.

Accordingly, in one embodiment, the DNA binding domain (DBD) of theglucocorticoid receptor is replaced by that of another transactivator,in particular that of the yeast protein Gal 4 and the sequence forcontrol of transcription (RE) of the glucocorticoid receptor is replacedby that of another transactivator, in particular the 17 mer (17m)sequence recognized by the Gal4 protein.

The subject of the present invention is therefore also a method ofexpressing a foreign protein in human or animal, in particularmammalian, cells, characterized in that cells are cultured which containan expression vector system according to the invention, and in that saidsynthetic ligand is added to the culture medium and then the proteinsynthesized is recovered.

The synthetic glucocorticoid ligands diffuse freely in the cells whenthey added to the culture medium or injected into an animal.

In this method, a synthetic ligand chosen from dexamethasone,triamcinolone acetonide, RU2836, bimetrazole, deacylcortivazol andfluocinolone acetonide will be used in particular.

The subject of the present invention is, in addition, a fusion proteincomprising a receptor according to the invention or a ligand bindingdomain according to the invention and a protein whose activity is morestrongly inducible by binding of said receptor or of said ligand bindingdomain (LBD) of said receptor with a said synthetic glucocorticoidligand than with a natural glucocorticoid ligand.

In particular, the subject of the present invention is a fusion proteinwhich comprises a recombinase protein and more particularly of thefamily of λ integrases and still more particularly the Cre protein ofthe bacteriophage P₁.

In one embodiment, the fusion protein comprises the C-terminal part ofthe hinge region D of the human nuclear glucocorticoid receptor,intercalated between the Cre protein and said ligand binding domain ofthe modified receptor according to the invention.

The actual recombinase activity of the fusion protein is similar to thatof Cre. It therefore makes it possible, like Cre, to excise or reversethe DNA sequences located between the loxP sites, to insert a plasmidcontaining a loxP site at the level of a loxP site located in thegenome, or to carry out a translocation between two chromosomes eachcontaining a loxP site.

The subject of the present invention is also a fusion gene coding forthe fusion protein according to the invention comprising a DNA fragmentcoding for a modified nuclear glucocorticoid receptor or a ligandbinding domain of said receptor according to the invention, as well as aDNA fragment coding for a protein of which it is desired to regulate theactivity, said activity being inducible by binding of said receptor orof said ligand binding domain with a said synthetic glucocorticoidligand, but not by binding with a natural glucocorticoid ligand whensaid protein is fused to said receptor or to said ligand binding domainof said receptor.

More particularly, the subject of the present invention is a fusion genecomprising, in the 5′→3′ direction:

a DNA fragment coding for the Cre recobinase of bacteriophage P₁:

a DNA fragment coding for all or part of the hinge region D of thenuclear glucocorticoid receptor, a region situated between the DBDdomain and the LBD domain, and

the DNA fragment coding for the modified, in particular mutated, ligandbinding domain (LBD) of the nuclear glucocorticoid receptor according tothe invention.

And still more particularly, the subject of the present invention is afusion gene, characterized in that it has, as sequence, a sequencecoding for the amino acid sequence of SEQ ID NO: 2 comprising:

amino acids 1 to 343 which correspond to the Cre recombination;

amino acids 346 to 377 which correspond to the C-terminal region of theD region of the human glucocorticoid receptor;

amino acids 378 to 623 which correspond to the LBD [GR(I747/T)].

In a specific embodiment, the fusion gene has substantially, assequence, the Cre-LBD[GR(I747/T)] sequence as represented in SEQ ID NO:2 in which amino acids 626 to 667 correspond to the F region of thehuman estrogen receptor.

The subject of the present invention is also a vector for expressing thefusion protein encoded by the fusion gene according to the invention, inanimal, in particular mammalian, host cells, characterized in that itcomprises the fusion gene according to the invention, placed under thecontrol of elements for expression ensuring its expression in said hostcells. The vector for expressing the fusion protein may also be a vectoror integrating into the genome of the host cells.

In the vectors according to the invention, to express the receptor, theLBD or the fusion protein according to the invention, it is necessaryfor the DNA fragments coding for these molecules to be placed under thecontrol of a regulatory sequence, in particular of the promoter/enhancersequences. Promoter/enhancer sequences of the SV₄₀ virus may be used inparticular.

The subject of the present invention is, in addition, a method ofrecombination, in particular of conditional excision, insertion,inversion or translocation at the level of a DNA fragment containing oneor two sites for specific recognition of a recombinase protein in humanor animal, in particular mammalian, host cells, characterized in that itcomprises the steps in which:

1) a fusion protein according to the invention or a vector forexpressing said fusion protein according to the invention is introducedinto said host cells under conditions allowing the expression of thefusion protein according to the invention and,

2) said fusion protein is complexed with a said synthetic glucocorticoidligand by exposing said ligand to said host cells.

The present invention therefore provides a method for the conditionaldeletion of a DNA fragment in which a method of excision according tothe invention is used, and in which said DNA fragment to be excised isintegrated between two recombinase protein recognition sites oriented asa direct repeat. In particular, said DNA fragment may be chosen so thatthe excision of said DNA fragment has, as effect, the inactivation ofsaid gene. The excision of said DNA fragment can also allow thesynthesis of a functional protein if said fragment contains, forexample, a stop codon or a polyadenylation signal.

In the preferred embodiment according to the invention, said recombinaseprotein specific recognition sites are the loxP sites and saidrecombinase protein is the Cre protein of bacteriophage P₁.

Said DNA fragment which it is desired to recombine, in particular toexcise and said recognition site(s) specific for a recombinase proteinmay be carried by a plasmid or viral vector, or may be integrated intothe chromosome of the host cells.

The integration of a gene carrying the loxP sites in a genome may berandom or targeted, In particular, the integration of the recombinaseprotein specific recognition sites, in particular of the loxP site(s)for the Cre recombinase, may take place by homologous recombination ofthe gene comprising said DNA fragment to be excised or inverted (2 loxPsites) or respectively inserted or translocated (1 loxP site) with asaid modified gene comprising said DNA fragment to be excised flanked in5′ and/or 3′ by said recombinase recognition site(s) depending on thedesired application, in particular the loxP sites.

A chimeric protein, Cre-LBD[GR(747/T)] of SID No. 2, composed of the Crerecombinase fused to the LBD of GR (I747/T) was constructed. The fusionprotein Cre-LBD[GR(I747/T)] makes it possible to carry out arecombination between the loxP sites, in a mammalian cell, following atreatment with synthetic glucocorticoids. In the absence of treatment,or in the presence of concentrations of natural glucocorticoids of up to10⁻⁶ M, no excision is observed. This chimera is capable of excising theDNA sequences situated between the loxP sites previously integrated intothe genome of F9 cells (mouse embryo carcinoma) or present on a plasmidafter transfection of these cells with a vector expressingCre-LBD[GR(I747/T)] following a treatment with Dex at 10⁻⁶ M or 10⁻⁷ M,whereas at such concentrations, cortisol does not induce recombinaseactivity. As already stated, the Cre-LBD[GR(I747/T)] expression vectormay also be a vector for integration into the aenome of the host cells.

This system therefore makes it possible to release the recombinaseactivity of a chimeric protein at given and chosen time. The fusionprotein Cre-LTD[GR(I747/T)] may be expressed in cells containing loxPsites without modifying the locus containing the loxP sites.Recombination at the level of the loxP sites takes place only aftertreatment with synthetic glucocorticoids. Furthermore, by expressing thefusion protein Cre-LBD[GR(I747/T)] in an animal under the control of apromoter exhibiting cellular specificity, it is possible to obtainrecombination between loxP sites, specifically in these cells.

The subject of the present invention is also a vector for transferring aDNA fragment into human or animal, in particular mammalian, host cells,characterized in that it comprises a said DNA fragment to be transferredcomprising recognition sites specific for the recombinase, in particulartwo loxP sites, oriented as direct repeats and a cassette for expressionof a fusion gene according to the invention. Appropriately, the twosites are placed at each end of said DNA fragment.

Preferably, the fusion gene is placed under the control of expressionelements specific for the host cells; in particular, it comprises thesequence of the plasmid pCre-LBD[GR(I747/T)] of SEQ ID NO: 3.

In the excision method according to the invention, said DNA fragment tobe excised may be transferred into the host cells before, at the sametime, or after the step of introducing the fusion protein or a transfervector.

The transfer vector according to the invention may be a plasmid or aviral vector. The mode of transfer varies according to the type oftarget cell and the desired efficiency.

When the material is transferred into the germ cells, microinjection ofthe male pronucleus of a fertilized oocyte is preferably used. Tointroduce the DNA into pluripotent embryonic cells (ES cells), theappropriate technique will instead be electroporation or the use ofretroviral vectors.

For experiments on somatic cells, it is sought to obtain a maximumefficiency, hence the preferential use of viral vectors: mainlyretroviruses and adenoviruses. These viruses should be defective, inparticular for application in human health to prevent any multiplicationor any risk of revertion.

After integration or otherwise of this type of vectors into the genome,the Cre-lox system makes it possible to excise certain viral sequenceswhich might possibly present a risk in relation to the subsequentpropagation of the virus. This is very advantageous for the use of suchvectors in gene therapy. Vectors containing a cassette for expression ofthe Cre-LBD[GR(I747/T)] gene and lox sites delimiting a potentially“dangerous” gene and this cassette make it possible, where appropriateafter integration into the genome of the recombinant virus, to eliminatethese elements. Conversely, it is also possible to activate a gene whichhas been integrated in an inactive form. This activation may result fromthe deletion of a fragment from said gene, for example a stop codon or apolyadenylation signal delimited by loxP sites.

When it is desired to transfer genetic material into a cell, constructsare generally used which comprise the gene to be transferred, to which ahelper gene has optionally been added, conferring a selective advantage(for example the neo gene conferring resistance to the antibiotic G418).

Selectable markers may also be excised by the excision method accordingto the invention.

By using conventional techniques, it is possible to modify, byhomologous recombination, mammalian, in particular mouse, genes. A loxPsite may be introduced into a gene which it is desired to modify, or twoloxP sites may delimit sequences which it is desired to modify. Inparticular, the selectable marker used to make it possible to identifythe homologous recombination events may be problematic, and may beremoved in a first instance, if necessary, if it is itself delimited byrecombinase recognition sites such as loxP or FRT sites, bymicroinjecting the corresponding recombinase protein, by transfectingthe cells, in particular ES cells, with an expression vector for arecombinase protein, or by crossing mice carrying the selectable markerwith mice expressing the desired recombinase, in particular the Crerecombinase. This makes it possible to obtain mice whose solemodification in the modified locus is the insertion of recognition sitessuch as loxP. These mice may subsequently be crossed with miceexpressing Cre-LBD[GR(I747/T)] and the modification of the locusobtained by treatment with the ligand such as Dex. This makes itpossible to inactivate or modify a gene at a given time, and thereforeto study the function of these genes at various times of development.This is particularly advantageous for studying the genes which areessential for the good progress of embryonic development. Indeed, insome cases, conventional homologous recombination techniques cause thedeath of the embryo and do not allow the function of the gene to bestudied at later stages.

The transfer of genes into a given cell forms the basis of gene therapy.The most efficient vectors in this regard are viral vectors, inparticular retroviral or adenoviral vectors (39). The subject of thepresent invention is therefore also a transfer vector according to theinvention comprising said DNA fragment to be transferred comprising twoloxP sites oriented as a direct repeat, for use as a medicament in genetherapy, when it is administered in combination with a said syntheticglucocorticoid ligand such as dexamethasone.

This type of vector makes it possible to transfer the desired DNAfragment comprising two recombinase specific recognition sites into agene so that it will be possible for the DNA sequences situated betweenthe two loxP sites to be conditionally excised after administration ofsaid synthetic glucocorticoid

The present invention provides, if addition, human or animal, inparticular mammalian, cells transfected with a vector for expression andtransfer of a fusion protein according to the invention, or human oranimal, in particular mammalian, cells into which a DNA fragment hasbeen transferred with the aid of a transfer vector according to theinvention, or alternatively human or animal, in particular mammalian,cells constitutively expressing the fusion protein according to theinvention.

A further subject of the present invention is finally a transgenicanimal, in particular a mouse, comprising a functional gene for thefusion protein according to the invention, said gene being in particularintegrated into one of its chromosomes.

Alternatively, a transgenic animal comprising a functional gene for thefusion protein according to the invention, in which a gene of interestis modified by insertion of loxP site(s), in particular integrated intoone or more chromosomes.

The subject of the present invention is finally the use of an expressionor transfer vector of cells or of an animal according to the inventionas a tool for analyzing or studying the function of a given gene of ahost cell and a method of treating cells ex vivo or in vitro involvingthe use of a method of excision, insertion, inversion or translocationaccording to the invention and the use of an expression or transfervector according to the invention.

Other characteristics and advantages of the present invention willemerge in the light of the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

SEQ ID NO: 1 represents the nucleotide and peptide sequences of GR(I747/T) with the DBD corresponding to aa 421-487 and the LBDcorresponding to aa 532-777;

SEQ ID NO: 2 represents the nucleotide and peptide sequences ofCre-LBD[GR(I747/T)].

Nucleotides 1-1029 code for the Cre recombinase (aa 1-343).

Nucleotides 1036-1131 code for the C-terminal region of the D region orthe human glucocorticoid receptor [aa 500-531 of GR (I747/T); aa 346-377of Cre-LBD[GR(I747/T)].

Nucleotides 1132-1869 code for the LBD of GR (I747/T) [aa 532-777 of GR(I747/T); aa 378-623 of Cre-LBD[GR(I747/T)].

Nucleotides 1876-2001 code for the F region of the human estrogenreceptor [aa 554-595 of HEGO; aa 626-667 of Cre-LBD[GR(I747/T)].

Nucleotides 1777-1779 correspond to the mutation which replacesisoleucine with a threonine.

SEQ ID NO: 3 represents the nucleotide sequence of the plasmidpCre-LBD[GR(I747/T)].

SEQ ID NOS: 4 to 17 represent the nucleotide sequences of the variousoligonucleotides used according to the invention.

FIG. 1 [SEQ ID NOS.: 20-21] is a schematic representation of Crerecombinase activities.

FIG. 2 represents the transcriptional activity of the mutant andwild-type GR receptor in response to dexamethasone and the capacity forattachment of dexamethasone to the two receptors.

FIG. 3 represents a Scatchard analysis of the binding of[³H]-dexamethasone to the wild-type and I747/T mutant receptors.

FIG. 4 represents competition by RU 486 for the Dex binding and thetransactivation activities of the wild-type (WT) and GR (I747/T)receptor.

FIG. 5 is a schematic representation of the Cre, hER, Cre-ER, hGR andCre-LBD[GR(I747/T)] proteins.

FIG. 6 is a schematic representation of the plasmidpCre-LBD[GR(!747/T)].

FIGS. 7A-D represents the nucleotide sequence ofpCre-LBD[GF(I747/T)](SEQ ID NO: 5) and the peptide sequence ofCre-LBD[GF(I747/T)] (SEQ ID NO. 5.

FIG. 8 represents the PCR strategy used to detect the wild-type, mutatedand recombinant alleles in the test for recombinase activity ofCre-LBD[GR(I747/T)].

FIG. 9 represents the PCR analysis of the excision of the sequencessituated between the loxP sites after transient transfection ofpCre-LBD[GR(I747/T)] in the murine cells RXRα^(+/−(LNL)) withoutaddition of ligand, or treated with cortisol (10⁻⁶ M) or withdexamethasone (10⁻⁶ M).

FIG. 10 represents the PCR analysis of the recombinase activity ofCre-GR(I747/T) in the cells RXRα^(+/−(LNL)):Cre-GR(I747/T) not treatedor treated with cortisol or dexamethasone at 10⁻⁶ M.

FIG. 11 summarizes the recombinase activity of Cre-LBD[GR(I747/T)] inthe RXRα^(+/−(LNL)) line constitutively expressing the recombinase, inthe presence of increasing concentrations of various ligands.

FIG. 12 represents the PCR analysis of the recombinase activity ofCre-LBD[GR(I747/T)] induced by synthetic ligands in spite of thepresence of natural ligands.

FIG. 13 represents the test for the recombinase activity ofCre-LBD[GR(I747/T)] in human HeLa cells.

FIG. 14 is a schematic representation of the trangeneCre-LBD[GR(I747/T)]. Part (a) represents the DNA fragment containing thesequence coding for the Cre-LBD[GR(I747/T) ] gene used to establishtransgenic mice and part (b) represents the genomic structure of thewild-type allele RXRα, of the target allele RXRα^(ΔAF1 (LNL)) and of theexcised allele RXRα^(ΔAF1 (L)) as well as the PCR strategy for analyzingthe excision of the marker.

FIG. 15 represents the PCR analysls of the recombinase activity ofCre-LBD[GR(I747/T)] in mice treated with dexamethasone.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Replacement of isoleucine 747 with threonine in the human nuclearglucocorticoid receptor.

A human GR mutant was obtained comprising a single amino acidreplacement, that of the isoleucine 747 residue by a threonine residue,located between helices H11 and H12, in the C-terminal part of theligand binding domain. This mutation appeared spontaneously in thevectors derived from HG1 (40). This mutant is different from other GRLBD mutants previously described (22, 38), in which the alteredactivation function is closely correlated with a modification in theirligand binding properties in vitro.

1. Mutation in the Human GR

The vector for expression of the wild-type human GR (pSG5HG0) wasobtained by cloning the BamHI fragment of about 2.9 kb (containing theopen reading frame of the wild-type hGR) isolated from the HG0 vectorpreviously described (40), into the BamHI site of the expression vectorpSG5 (41).

All the DNA constructions were performed by means of conventionaltechniques (42). The expression vector GR (I747/T)hGR, coding for amutant carrying the I 747→T mutation, was obtained from the fragment ofPstI-PflMI digestion of the mutated expression vector HG1 (40) which wasligated into the corresponding PstI and PflMI sites of pSG5HG0.

In addition to the isoleucine 747 residue to a threonine residuemutation present in HG1, this mutant also contains a mutation of theEcoRI site which does not result in any alteration of the peptidesequence.

The presence of the mutation was confirmed by sequencing (Sequenase 2.0protocol, U.S. Biochemical Corp., Cleveland, Ohio).

2. Results

2.1. The Replacement of Isoleucine 747 by Threonine Leads to Shift inthe Dexamethasone Dose-response Curve for Transactivation, WithoutSubstantially Affecting the Ligand Binding Affinity.

The transcriptional characteristics of the wild-type (WT) hGR and of amutant in which isoleucine 747 was replaced by threonine [designatedhereinafter GR (I747/T)] were compared. The receptors were transientlyexpressed in GR-free CV-1 cells, conjointly with a luciferase reportergene under the control of the tyrosine aminotransferase promoter(pTAT-tk-Luc). FIG. 2 shows the corresponding curves of response as afunction of ligand dose used, allowing the EC₅₀ (ligand concentrationgiving 50% of the maximum transcriptional response) values to bedetermined. The dose-response curve for WT hGR exhibits a maximumresponse at 10⁻⁸ M Dex and a response equal to half the maximum responseat 4×10⁻⁸ M, whereas the transcriptional response curve for the GR(I747/T) mutant is markedly shifted toward the highest ligandconcentrations, resulting in 100-fold higher values of the EC₅₀ for Dex(4×10⁻⁸ M) (see Table I below). The maximum stimulation mediated by GR(I747/T) was reached at about 10⁻⁶ M Dex. However, it is important tonote that the GR (I747/T) receptor is capable of activating thetranscription of pTAT-tk-Luc to the same degree as the WT GR.Consequently, the replacement of the isoleucine residue by threonine atposition 747 does not reduce the ability of GR to activate transcriptionbut specifically modified the ligand sensitivity of the response.

The effect of this mutation is independent of the N-terminal A/B regionand of its associated transactivation function AF-1. The deletionmutants lacking AF-1 but containing the I747/T[ΔA/B-GR (I747/T)]mutation require 3×10⁻⁸ M Dex for a stimulation representing half themaximum stimulation, whereas only 6×10⁻¹⁰ M are sufficient for theΔA/B-GR derived from the WT (data not represented; see also Table II forthe corresponding GAL chimeras). Thus, the I747/T mutation exerts asimilar effect on the complete GCr and the CR with N-terminal deletion.It can be concluded therefrom that the shift observed in the response asa function of the ligand and dose is not the consequence of modifiedinteraction between AF-1 land AF-2.

To determine the effect of the I747/T mutation on the binding of theligand, the binding of Dex was measured in a cytosol of Cos-7 cellstransiently expressing the mutant hGR and the wild-type hGR. The K_(d)values obtained from a Scatchard analysis are very similar since theI747/T mutant only manifests an affinity for Dex which is twice as lowas the WT GR (FIG. 3). Thus, the order of magnitude of the shift in thedose-response curves toward the high concentrations of hGR (I747/T),compared with the WT hGR (about 100 fold) is not very likely to resultfrom a decrease in affinity for the ligand for the mutated hGR (factorof 2 only; compare FIGS. 2 and 3).

2.2. hGR (I747/T) Does Not Respond to the Natural Ligands

To further characterize the binding characteristics of the I747/Tmutant, the activation by various known synthetic agonists for the WT GR(triamcinolone acetonide, RU28362, bimedrazol), a partial agonist(fluocinolone acetonide) and natural glucocorticoids (cortisol,corticosterone), was compared. With the mutant receptor, triamcinoloneacetonide and RU28362 give the same transactivation as with thewild-type receptor, whereas bimedrazol and fluocinolone acetonidemanifested a higher activity (Table I). For all these analogs, theI747/T mutant manifests a shift in the dose-response curve in thedirection of the higher ligand concentrations, although this shift isless pronounced for high affinity ligands (Table 1).

Natural ligands, such as cortisol or corticosterone or aldosterone, thenatural ligand for the mineralocorticoid receptor, bind to GR with alower affinity than Dex. With the wild-type GR, these ligands are aseffective as Dex for stimulating the luciferase reporter (Table I).However, in the presence of these natural ligands, and in notablecontrast with the effects observed with Dex, transactivation by theI747/T mutant is greatly reduced (cotisol) or suppressed(corticosterone, aldosterone).

2.3. Similar Agonist and Antagonist Activities of RU486 With Wild-typehGR and Mutant hGR

RU486 is a potent inhibitor of the expression of genes which aredependent on glucocorticoids. In the presence of this antagonist, theAF-2 function of GR is blocked, but it has been possible to observe someAF-1-dependent agonist activity [(43) and references contained therein].A mutation or a deletion of the C-terminal part of the LBD can alter theresponse to agonist/antagonist ligands, although the roles of AF-1 andAF-2 have not been clearly defined in the studies reported (44-46).

To test the relative binding affinity of RU486 for the I747/T mutant andthe wild-type, the ability of RU486 to enter into competition with Dexfor binding to the receptors was examined. Cells transfected with mutantor WT hGR were treated with 10 nM [³H]-Dex, a concentration sufficientto generate essentially mutant and WT receptors complexed with Dex, inconformity with the in vitro ligand binding tests (see above), and withincreasing concentrations of RU486 (FIG. 4). Very similar IC₅₀ (theantagonist concentration blocking 50% of the maximum transactivation)values were obtained for the two receptors (10⁻⁸ M for the WT hGR and6×10⁻⁹M for the mutant hGR). These results are in agreement with thetests for binding to Dex and show that the affinities for binding to Dexand RU486 are not modified by the I747/T mutation.

2.4. The Shift in the Transcriptional Activity of the Mutant Receptor isIndependent of the Promoter

The results described above were obtained in transfected cells using areporter gene containing a promoter fragment of the TAT gene whichharbors two glucocorticoid response elements (GRE). To distinguishbetween a modification of the intrinsic transactivation properties ofthe receptor and modification of the cooperative activity resulting fromthe binding of several receptor molecules, the transcriptionalactivities of the mutant receptor GR (I747/T) and of the wild-typereceptor were compared using a synthetic promoter containing a singleGRE, which is palindromic and perfect. The corresponding dose-responsecurves have the same profiles as those obtained with the TAT promoter.The response corresponding to half the maximum response is obtained at10⁻¹⁰ M for the WT GR and at 2×10⁻⁸ M for GR (I747/T) (Table II). Thisindicates that the shift observed with the mutant receptor reflects theintrinsic transactivation properties of the mutant GR (I747/T) and arenot the result of an alteration in the cooperation between thereceptors.

The replacement of the DNA binding domain of GR by the first 147 aminoacids of the yeast Gal4 transactivator gives a hybrid activator proteinhaving a new promoter specificity. The Gal4-GR hybrids consist of theDNA binding domain (DBD) of Gal4(1-147) fused with the LBD of WT ormutant hGR. Their capacities to activate transcription were tested intransient transfection tests in HeLa cells. The same shift was observedas with the complete GR (I747/T) mutant (Table II). These results showthat the shift in the transcriptional response as a function of theligand dose is independent of promoter and of response element (RE). Inaddition, this effect is apparently not specific for the strains ofcells used, as has been observed both in human HeLa cells and in simianCos cells. To further exclude a possible involvement of factors bindingto the tk or β-globin promoter of the reporter gene (47), a reportergene was used containing a Gal4 response element preceding a minimalpromoter. Although transactivation by the Gal4 chimeric protein wasreduced with this minimal reporter gene, it was still possible toobserve a shift in the response curve toward the increasing doses forthe mutant chimeric protein Gal4-GR (I747/T) (Table II).

2.5. Transcriptional Activity of Mutant and Wild-type GR Receptors inResponse to Dexamethasone (FIG. 2).

CV-1 cells were transiently transfected with 0.5 μg of vectors forexpression of wild-type GR (□) or of GR (I747/T) (o), 2.5 μg ofpTAT-tk-Luc as reporter plasmid and 0.5 μg of pCMV-βGal as internalcontrol. After transfection, the cells were incubated for 24 hours withincreasing concentrations of Dex, and they were then subjected to anassay of the β-galactosidase and luciferase activities, as described in“Materials and Methods”. The luciferase activity was normalized with theβ-galactosidase activity, and the value obtained in the absence ofligand was subtracted for each point. The results were expressed as apercentage of the maximum luciferase activity obtained for eachreceptor. Each point is the mean±standard deviation of the mean of fourseparate determinations carried out in duplicate. The analyses ofbinding at saturation were carried out on cytosols obtained from Cos-7cells transfected with 40 μg of vector for expression of wild-type hGR(□) or of hGR (I747/T) (•), with 20 μg of pCMV-βGal incubated overnightat 4° C. in the presence of increasing concentrations of[³H]-dexamethasone±a 100× excess of radio-inert Dex. The [³H] steroidattached was measured after treatment with dextran-coated charcoal. Theresults presented are those for a single assay carried out in duplicateand representative of three separate assays. The saturation curves forthe binding of [³H]-dexamethasone are represented as a percentage of themaximum binding capacity obtained for each receptor. The bindingcapacity of the GR (I747/T) mutant is reduced to reach about 40-60% ofthat of the wild-type hGR (623±65 fmol/mg of protein, compared with1340±285 fmol/mg of protein).

2.6. Scatchard Analysis of the Binding of [³H-dexamethasone to theWild-type and I747/T Mutant Receptors (FIG. 3).

The Scatchard analysis was applied to the binding performed as describedin FIG. 2. The apparent dissociation constant for the GR (T747/T) mutant(o) is only twice as high as that for the wild-type hGR (υ)(K_(o)=5.1±1.9 compared with 2.7±1.3 nM).

TABLE I 2.7. Transcriptional activities and EC₅₀ of various steroidswith wild-type hGR and hGR(I747/T) Luciferase activity % EC50 Wild hGRhGR Ligand type (I747/T) Wild type (I747/T) % EC₅₀ Dexamethasone 100 1004 × 10⁻¹⁰M 4 × 10⁻⁸M 100 TA 98 102 5 × 10⁻¹⁰M 2 × 10⁻⁸M 40 RU28362 90 883 × 10⁻¹⁰M 7 × 10⁻⁹M 23 Fluocinolone 75 100 1.5 × 10⁻¹⁰M 3 × 10⁻⁹M 20Bimedrazol 100 180 10⁻¹⁰M 2 × 10⁻⁹M 20 Cortisol 110 15 7 × 10⁻⁹M nmCorticosterone 100 0 7 × 10⁻⁸M nm Aldosterone 90 0 5 × 10⁻⁸M nm

Co-transfections were carried out and dose-response curves wereconstructed as described in FIG. 2. Six increasing concentrations weretested, in duplicate, triamcinolone acetonide (TA), RU28362,fluocinolone acetonide (fluocinolone), cortisol, corticosterone andaldosterone. The assays of luciferase and β-galactosidase activity werecarried out as described in “Materials and Methods”. The mean values ofthree independent assays are given in this table. nm=not measurable.

2.8. Competition for the Dex Binding and for the TransactivationActivities by RU486 (FIG. 4).

Cos-7 cells transfected with the wild-type hGR (□) or I747T mutant (e)were incubated for 1 hour at 37° C. with 10 nM [3H]-dexamethasone aloneor in the presence of the indicated concentration of nonlabeled RU486,as descibed in “Materials and Methods”. The data are expressed as apercentage of the specific binding observed the control not subjected tothe competition. These data represent the mean of determinations carriedout in duplicate for each antagonist concentration.

The antagonist activity was determined on CV-1 cells transfected asdescribed in FIG. 4 and treated simultaneously with 10⁻⁷ M Dex and theindicated concentration of RU486, 24 hours before harvesting for theassay of the luciferase and β-galactosidase activities. 100% activity isdefined for each receptor as the luciferase activity of the cellstreated with Dex alone. Each point represents the mean of at least twoindependent tests carried out in duplicate. (□) wild-type hGR; (o):I747T mutant.

TABLE II 2.9. EC₅₀ obtained with various reporter genes ReceptorReporter gene EC₅₀ Wild-type GR PGRE-tk-Luc 10⁻¹⁰M GR(I747/T) ″   2 ×10⁻⁸M Gal4-GR p(17M)₅-βGlob-Luc 1.7 × 10⁻⁹M Gal4-GR_((I747/T)) ″ 1.5 ×10⁻⁷M Gal4-GR p(17M)₅-tata-Luc   2 × 10⁻⁹M Gal4-GR_((I747/T)) ″   2 ×10⁻⁷M

HeLa cells (for the Gall chimeras) or CV-1 cells were transientlycotransfected with various expression vectors and reporter genes. Thetransfection efficiency was standardized by cotransfecting with 0.5 μgof vector pCMV-βgal. The cells were treated for 24 hours with increasingconcentrations of dexamethasone. The luciferase and β-galactosidaseassays were carried out as described in “Materials and Methods”. Theluciferase activity is expressed as a percentage of the maximum activityobtained for each receptor and each reporter gene. The EC₅₀ values weredetermined graphically.

2.10. Outline

The isoleucine 747 mutation of the human glucocorticoid receptor tothreonine results in a very high reduction, or even in an abolition, ofthe transactivation activity of the receptor, in the presence of naturalglucocorticoids, whereas at similar concentrations, syntheticglucocorticoids efficiently stimulate the transactivation activity ofthis mutated receptor.

It is important to underline that hGR (I747/T) responds fully to a highconcentration of Dex, which indicates that it can, in principle,transactivate as efficiently as its wild-type counterpart. Consequently,the transcriptional activation function of AF-2 itself is apparently notaltered by the mutation, as is the case for receptors comprisingmutations in the core part of AF-2 (see the introduction for thereferences).

3. Materials and Methods

3.1. Cell Cultures and Transfection

The CV-1 and Cos-7 cells were cultured in Dulbeco's modified Eagle'smedium (DMEM) containing 10% (v/v) of fetal calf serum (FCS) (Gibco,Grand Island, N.Y.), in a humidified atmosphere containing 5% CO₂. TheHeLa cells were cultured under the same conditions. Transienttransfections were carried out in 6-well multiplates according to thecalcium phosphate procedure already described (42). A referencerecombinant plasmid, pCMV-βgal, expressing bacterial β-galactosidase,was transfected conjointly with the receptor expression vector and thecorresponding reporter gene so as to correct possible variations in hetransfection efficiency. The precipitate was removed by washing after 24hours, and the cells were kept in DMEX without FCS but containinghormone. After another 24 hours, the cells were rinsed and lysed for 15minutes with 0.3 ml of lysis buffer [25 mM Tris.phosphate, pH 7.8, 8 mMMgCl₁, 1% Triton X100, 10% glycerol (48)]. The corresponding luciferaseactivity as determined on an aliquot fraction (0.1 ml), by evaluation ofthe luminescence peak (for an integration time of 15 seconds), afterinjection of 0.1 ml of 1 mM luciferin in an LKB luminometer. Theβ-galactosidase was determined by the method modified by Bocquel et al.(49), for controlling the efficiency of each transfection.

3.2. Plasmids Used

The N-terminal deletion GRs, ΔA/B-GR or ΔA/B-GR5I747/T), which expressedonly amino acids 368 to 777, were obtained from the fragment of PstI-PflMI digestion of the expression vector HG0 or pGR (I747/T), respectively,ligated into the corresponding sites of HG8 (49). The plasmid pGAL-GR(gift from T. Lerouge) consisted of the DNA binding domain of the yeastGal4 protein (aa1-147) and of the hormone binding domain of GR(a500-777); pGal-GR (I747/T) was constructed by insertion of themutation into pGal-GR.

The reporter gene pTAT-tk-Luc was provided by D. Gagne and wasconstructed as follows: an intermediate plasmid pTAT was first generatedby insertion of the 966 bp EcoRI-BamHI segment obtained from pKT531[offered freely by T. Grande (50)] into the corresponding sites of thevector pUC19 (Pharmacia). The fragment containing the sequence codingfor the firefly luciferase and the thymidine kinase (tk) minimalpromoter obtained from the Herpes simplex virus was isolated in the formof a BamHI fragment from pvit-tk-Luc (51) and inserted, downstream ofthe TAT promoter, into the corresponding site of pTAT. The reporterplasmid pGRE-tk-Luc was constructed by ligation of the fragment ofHindIII-BglII digestion of pGRE-tk-CAT (52), which contains a syntheticGRE (AGAACAcagTGTTCT) upstream of the tk minimal promoter, into theHindIII-BglII sites of pLuc (53). The reporter gene p(17M)₅-βGlob-Luchas been previously described (54). The reporter plasmidp(17M)₅-tata-Luc was constructed from the plasmid p(17M)₅-tata-CAT (55)by digestion with XhoI-BamHI and isolation of the fragment containing(17M₅)-tata. This fragment was then inserted into the vector pGL₂(Promega) digested with the same restriction enzymes.

The β-galactosidase expression vector pCMV-βgal contains the sequencecoding for β-galactosidase, under the control of the strong constitutivecytomegalovirus (CMV) promoter.

3.3. Test of Binding to a Hormone

Cos-7 cells transfected with pCMV-βgal and wild-type hGR or I747/Tmutant were harvested 48 hours after the transfection, in aphosphate-buffered salt solution (STP), by scraping with the aid of arubber spatula. The cells were washed twice in ice-cold STP. All thesteps for the preparation of cytosols were carried out at 4° C. Thecells were resuspended in 3 volumes of binding buffer (20 mM Na-HEPES,pH 7.3, 20 mM sodium molybdate and 5 mM EDTA (22)] and with proteaseinhibitors [leucopeptin (5 μg/ml), aprotinin (5 μg/ml), PMSF (40 μg/ml)and pepstatin A (5 μg/ml)], and they were placed for 15 minutes on ice.The cells were then lysed by 30-40 piston strokes in a Dounce glasshomogenizer, and the resulting homogenate was then centrifuged at110,000 g for 30 minutes. The supernatant, called cytosol, was usedimmediately for the test of binding to a hormone. The proteinconcentrations were determined by the Bio-Rad protein assay (Bio-RadLaboratories, Hercules, Calif., USA). For the test of binding to ahormone, a cytosol, containing 1-3 mg of protein/ml, was incubatedovernight at 4° C. with [³H]-Dex (49 CI/mmol; Radiochemical Center,Amersham, England) or cortisol (67 Ci/mmol; Radiochemical Center,Amersham, England) without (total binding) or with (non-specificbinding) a 100-fold excess of radio-inert compound (10⁻⁵M, SigmaChemical Co., St. Louis, Mo., USA). The samples were then treated with 1volume of dextran-charcoal (5% charcoal, 0.5% dextran in the bindingsolution) for 15 minutes on ice, with constant stirring, and then theywere subjected to centrifugation for 10 minutes at 12,000 g. The [³H]ligand attached was measured in an aliquot portion of the supernatant,by scintillation counting. The equilibrium dissociation constant (Kd) ofthe receptor for dexamethasone or cortisol was determined by Scatchardanalysis.

For the competitive binding analysis, the cytosols were incubated with10 nM [³H]-Dex and various concentrations of nonradioactive competitivesteroids, under the same conditions as above. After treatment withdextran-charcoal and scintillation counting, the specific binding wasexpressed as a percentage of the control not subjected to competition,and plotted as a function of the concentration of steroid incompetition. Competitive binding tests in whole cells were carried outon 10⁶ cells incubated for 1 hour at 37° C. with 10 nM [³H]-Dex andvarious concentrations of steroids in competition. After sonication in 1ml of KCl buffer (1.5 mM EDTA, 20 mM Tris.HCl, pH 7.4, 0.5 M KCl), theunbound steroid was removed by addition of two volumes of dextran-coatedcharcoal, in KCl buffer with gelatin (0.05% dextran, 0.5% charcoal, 0.1%gelatin). The mixture was incubated for 30 minutes at 4° C. and it wascentrifuged at 3000 g for 5 minutes. The radioactivity was determined byliquid scintillation counting and the specific binding was plotted on acurve as above.

EXAMPLE 2

Chimeric Cre Recombinase Whose Activity is Inducible by SyntheticGlucocorticoids, but not by Natural Glucocorticoids

1. Materials and Methods

1.1. Construction of the Vector pCre-LBD[GR(I747/T)]

A ClaI-XbaI DNA fragment of about 1300 base pairs (bp), containing thesequence coding for the ligand binding domain of the humanglucocorticoid receptor was isolated from the plasmid HG1 (see Example 1(40)) and its ends were filled by T4 polymerase. This fragment wasmutated so that the nucleotides ATT coding for an isoleucine at position747 of the wild-type receptor (56) are replaced by the nucleotides ACC,coding for a threonine. This mutated fragment was cloned into the vectorpCre-ER (28) digested with XhoI and PstI whose ends were also filled byT4 polymerase [plasmid pCr⊕GR(I747/T)+F; coding sequence of the LBD ofhGR (I747/T) in the same orientation as that of Cre].pCre-LBD[GR(I747/T)] was obtained by site-directed mutagenesis carriedout on the single-stranded DNA prepared from the plasmid pCre+GR(I747/T)+F, with the synthetic oligonucleotides:

SG52 (5′-CTGGAAGATGGCGATCTCGAGATTCAGCAGGCCACT-3′) (SEQ ID NO: 6)

SG53 (5′-CTGTTTCATCAAAAGGGTACCAGCCGTGGAGGGGCAT-3′) (SEQ ID NO. 7)

making it possible to fuse, on the one hand, the DNA sequence coding forCre [amino acids (aa) 1-343 (orf2(cre); (31)], and that which codes forthe region containing the mutated LBD of HG1 [aa 500-777], on the otherhand, the previously modified DNA sequence and that coding for the Fregion of the human estrogen receptor (ER) [aa 553-595 (57)] (FIG. 5).This operation restores the XhoI restriction site situated at the end ofthe Cre sequences in pCre-ER and introduces a KpnI restriction sitebetween the sequences coding for GR and the F region of ER (FIGS. 6 and7).

1.2. Mutation of the RXRα Gene

The vector (pRXRα^((LNL))), used to mutate the RXRαgene (58) byhomologous recombination in F9 cells was constructed in the followingmanner:

A λ phage containing a genomic fragment of 11.8 kb, comprising exons 2,3 and 4 of RXRα, was isolated from a genomic library prepared with theP19 murine embryo carcinoma genomic DNA, cloned into the EMBL3λ vector.A SalI fragment isolated from this phage (whose ends were filled with T4polymerase) was subcloned into the BamHI site (after treatment with T4polymerase) of a derivative of the vector pBluescriptIIsK+ (Stratagene).In this derivative, the XhoI site has been eliminated. An XhoI site wasintroduced into exon 4 of RXRα at the level of the AccI sitecorresponding to nucleotide 535 of the cDNA for RXRα (the cDNA sequencefor RXR∝ is given in Genbank reference: M 84817), by cloning thefollowing synthetic oligonucleotides into the AccI site:

5′-ATTATTATTACTCGAGTGATGTG-3′ (SEQ ID NO: 8) and,

5′-ATCATCATCATCCGAGTAATAATA-3′ (SEQ ID NO: 9).

Finally, the XhoI fragment, containing the cassette for expression ofthe neomycin resistance gene, surrounded by loxP sites, isolated fromthe vector pHR56 (28) was cloned into the XhoI site of the precedingvector.

The mutation of an allele of the RXRα gene in F9 wells was carried outby homologous recombination with the aid of the vector pRXRα^((LNL))(28). This line was called RXRα^(+/−(LNL)).

1.3. Expression of Cre-LBD[GR(I747/T)] in Cells Comprising a ModifiedDNA Fragment: RXRα^((LNL))

Transient transfection: 5×10⁶ RXRα^(+/−(LNL)) cells or HeLa cellsresuspended in 500 μl of PBS were electroporated with 5 μg of plasmidpCre-LBD[GR(I747/T)] [optionally cotransfected with 5 μg of the vectorpRXRα^((LNL)) for the HeLa cells] with the aid of an electroporator(Bio-Rad gene pulser), set at 200 V and 960 μF. The cells were theninoculated at 10⁶ cells per 100 mm dish and treated, if necessary, withligands.

Stable transfection: 5×10⁶ RXRα^(+/−(LNL)) cells, resuspended in 500 μlof PBS were electroporated with 5 μg of pCre-LBD[GR(I747/T)] and 1 μg ofpPGK-Hyg (59), digested respectively with SalI and PvuII, with the aidof an electroporator (Bio-Rad gene pulser), set at 200 V and 960 μF. Theresistant clones were obtained according to the procedure described byMetzger et al. (28).

1.4. “PCR” Amplification of a Genomic DNA Region Located in Exon 4 ofRXRα

The DNA amplifications were carried out using the “Polymerase ChainReaction” (PCR) technique according to the protocol described by Chenand Evans (60). The primers used are: SB211 and PZ105, havingrespectively as nucleotide sequence 5′-GGCAAACACTATGG-3′ (SEQ ID NO: 10)and 5′-TTGCGTACTGTCCTCTT-3′ (SEQ ID NO: 11). After denaturation for 8min at 94° C., 2.5 units of Taq polymerase are added. The amplificationis carried out by performing 35 cycles (30 sec. at 94° C., 30 sec. at50° C.) followed by one cycle (30 sec. at 94° C., 30 sec. at 50° C., 5min at 72° C.), the product of which is stored at 4° C.

The products of the reactions were separated on polyacrylamide (10%) oragarose (2.5%) gel. The DNA was visualized by UV followed by ethidiumbromide staining, or by Southern blotting (42).

1.5. Test of Excision of the Sequences Situated Between the LoxP Sites

The DNA of RXRα^(+/−(LNL)) cells, transfected with pCre-LBD[GR(I747/T)]or the parental vector pSG5, not treated or treated with cortisol (10⁻⁶M) or dexamethasone (10⁻⁶ M) for 72 hours, was subjected to a PCRreaction according to the strategy described in FIG. 8. The products ofthe reaction were separated on acrylamide gel and stained with ethidiumbromide. Lane M in FIG. 9 corresponds to the loading of the pBR322 sizemarker digested with HinfI. The position of the wild-type andrecombinant alleles is indicated.

1.6. PCR Analysis of the Recombinase Activity of Cre-LBD[GR(I747/T)] inthe RXRα^(+/−(LNL)) Line Constitutively Expressing the Chimeric Protein

For FIG. 10, the DNA prepared from RXRα^(+/−(LNL)):Cre-GR(I747/T) cellsnot treated or treated with cortisol (10⁻⁶ M) or with dexamethasone(10⁻⁶ M) served as a template for carrying out a PCR amplification,according to the procedure described in FIG. 8. The amplified DNA wasseparated on acrylamide gel and stained with ethidium bromide. Lanes 1,2, 3 and 4 contain respectively the PCR products produced with DNAextracted from wild-type (WT) F9 cells, RXRα^(+/−(LNL)), RXRα^(+/−(L))cells and C2RXRα^(+(L)/−(L)) cells (73). Lane 8 contains a size marker(1 kb ladder; GibcoBRL).

In FIG. 11, the DNA prepared from RXRα^(+/−(LNL)):Cre-GR(I747/T) cellsnot treated or treated with aldosterone, cortisol, corticosterone,dexamethasone, triamcinolone acetonide or RU486, at concentrationsranging from 10⁻⁹ to 10⁻⁶ M, served as a template for carrying out a PCRamplification, according to the procedure described in FIG. 8. Theamplified DNA was separated on agarose gel and analyzed according to theSouthern technique. The amplified fragments corresponding to thewild-type and recombinant alleles were detected by hybridization withthe aid of the synthetic oligonucleotide (5′-AAGAAGCCCTTGCAGCCCTC-3′)(SEQ ID NO: 12), radiolabeled at the 5′ end with T4 polynucleotidekinase. The signal was visualited and quantified with the aid of a“phosphoimager” (Fujix bas2000).

For FIG. 12, RXR∝^(+/−(LNL)):Cre-GR(I747/T) cells were cultured for 72 hin culture medium to which three natural ligands (aldosterone, cortisoland corticosterone) at the concentration of 10⁻⁹ M or 10⁻⁸ M anddexamethasone (10⁻⁷ or 10⁻⁶ M) have been added as indicated. Therecombinant allele was detected according to the procedure described inFIG. 8. The amplified DNA was separated on agarose gel and analyzedaccording to the Southern technique. The amplified fragmentscorresponding to the recombinant allele were detected by autoradiographyfollowing hybridization with the synthetic oligonucleotide5′-AATTATAACTTCGTATAATGTATGCTATACGAAGTTATTC-3′ (SEQ ID NO: 13),(containing a loxP site) radiolabeled with Phosphorus³² at the 5′ end byT4 polynucleotide kinase.

For FIG. 13, HeLa cells were cotransfected with the plasmidspRXRα^((LNL)) and pCre-LBD[GR(I747/T)] and then cultured for 72 hourswith or without addition of the appropriate ligands, at theconcentration of 10⁻⁷ M. The DNA of these cells was then subjected toPCR amplification according to the strategy described. The products ofthe reaction were separated on acrylamide gel and stained with ethidiumbromide. Lane 1 corresponds to the loading of the 1 kb ladder sizemarker (GibcoBRL).

2. Results

In order to express a Cre recombinase whose activity is inducible bysynthetic ligands for the glucocorticoid receptor, but not by naturalglucocorticoid ligands at “physiological” concentrations, an expressionvector, pCre-LBD[GR(I747/T)], derived from the vector pCre-ER (28) byreplacing the DNA sequence coding for amino acids 344-618 containing theLBD of the estrogen receptor (ER), by that coding for amino acids500-777 of a mutated glucocorticoid receptor (GR) containing the mutatedbinding domain (LBD) of GR (I747/T), was constructed. The constructionof this plasmid led to the insertion of the sequence GGTACC, coding forthe aa Gly-Thr at position 624-625 of Cre-LBD[GR(I747/T)] (FIG. 7).

The recombinase activity of Cre-LBD[GR(I747/T)] was tested in a murinecell line RXRα^(+/−(LNL)). This line was obtained by integrating, byhomologous recombination, the TK-neo gene, delimited by loxP sites (as adirect repeat) (LNL), into exon 4 of an allele of the RXRα gene (28).The TK-neo gene is a chimeric gene allowing the expression of athymidine kinase/neomycin resistance gene fusion protein.Electroporation of this line with the vector pCre-LBD[GR(I747/T)] madeit possible to evaluate its recombinase activity without addition ofligand, or after addition of cortisol (natural ligand) or ofdexamethasone (FIG. 9) (Dex; synthetic ligand). The DNA of the cellsthus treated is indeed subjected to a PCR with syntheticoligonucleotides located in exon 4, one upstream of the AccI site, theother downstream of this site. The size of the PCR fragments obtainedwith this pair of oligonucleotides is 65 bp for the wild-type allele,3345 bp for the mutated allele (after integration of LNL) and 191 bp forthe recombinant allele (after excision of the sequences situated betweenthe two lox sites and of one of the lox sites, see FIG. 8). Followingtreatment of the cells transfected with dexamethasone (10⁻⁶ M), a 191 bpfragment is observed, reflecting the presence of sequences correspondingto the recombinant allele, whereas in the absence of treatment orfollowing a treatment with cortisol (10⁻⁶ M), no fragment of this sizeis observed. These results therefore show that the recombinase activityof the fusion protein studied may be induced by treating cellsexpressing this protein with a synthetic GR ligand such as dexamethasonebut not with a natural ligand such as cortisol, at the concentration of10⁻⁶ M.

An RXRα^(+/−(LNL)) line constitutively expressing Cre-LBD[GR(I747/T)]was also established by cointegrating the cassette for expression of theCre-LBD[GR(I747/T)] gene with a vector expressing the hygromycinresistance gene (RXRα^(+/−(LNL)):Cre-GR(I747/T); see “Materials andMethods”; data not presented). The recombinase activity was testedfollowing a treatment of these cells with Dex (10⁻⁶ M) for 72 hours.Analysis of the products of the PCRs carried out on the DNA of thesecells reveals a fragment of 191 bp corresponding to the recombinantallele alone after treatment with Dex (FIG. 10).

Thus, it is shown that Cre-LBD[GR(I747/T)] is inactive without additionof ligand or in the presence of cortisol at 10⁻⁶ M, but active in thepresence of Dex, when its expression cassette is integrated into thegenome.

The kinetics of recombinase action was also evaluated: its activity isdetectable from 12 h of treatment with dexamethasone at 10⁻⁶ M, but itis considerably stronger after 72 h of treatment (results notpresented).

The recombinase activity of Cre-LBD[GR(I747/T)] was also testedfollowing the addition of various concentrations of ligands. Norecombinase activity could be observed in theRXRα^(+/−(LNL)):Cre-GR(I747/T) cells treated with aldosterone, withcortisol or with corticosterone (ligand concentration ranging from 10⁻⁹to 10⁻⁶ M), whereas in the presence of dex, of triamcinolone acetonideor of RU486, a recombinase activity is observed from 10⁻⁹ M (FIG. 11).

It was also shown, with the aid of this cell line, that the recombinaseactivity of Cre-LBD[GR(I747/T)] may be induced by synthetic ligands evenwhen the cells are cultured in the presence of natural ligands(aldosterone, cortisol and corticosterone) at 10⁻⁹ or 10⁻⁸ M. Theexcision of sequences located between the loxP sites is obtainedfollowing a treatment of 72 h with dexamethasone at 10⁻⁷ M or 10⁻⁶ M(FIG. 12).

The recombinase activity of Cre-LBD[GR(I747/T)] was finally tested inhuman cells: HeLa cells were transiently cotransfected with the vectorspCre-LBD[GR(I747/T)] and pRXRα^((LNL)), and then cultured either withoutaddition of ligand, or in the presence of the ligands indicated in FIG.13. As in the F9 cells, a 191 bp band corresponding to the recombinantfragment is amplified from DNA isolated from the cells treated withsynthetic ligands (Dexamethasone, Triamcinolone acetonide or RU486 at10⁻⁷ M), whereas without addition of ligand, or in the presence ofnatural ligands such as aldosterone, corticosterone or cortisol at thesame concentrations, only the fragment corresponding to the wild-typeallele can be detected.

These results therefore show that the recombinase activity of the fusionprotein Cre-LBD[GR(I747/T)] is induced by synthetic ligands for theglucocorticoid receptor, whereas it is insensitive to the naturalligands such as cortisol, corticosterone or aldosterone, even atconcentrations as high as 10⁻⁶ M. Furthermore, the presence of naturalligands does not prevent the induction of the recombinase activity ofCre-LBD[GR(I747/T)] by the synthetic ligands.

EXAMPLE 3 Induction of the Activity of a Chimeric Cre Recombinase inMice 1. Materials and Methods

1.1. Construction of Plasmids and Establishment of Transgenic Mice

The vector pCMVCre-LBD[GR(I747/T)] was constructed by cloning the 2 kbEcoRI fragment isolated from pCre-LBD[GR(I747/T)] into the EcoRI site ofpMGSV1 (60). The 4.64 kb PvuII fragment isolated frompCMVCre-LBD[GR(I747/T)] was injected into F1 zygotes (C57BL/6×SJL) at aconcentration of 4 ng/μl so as to produce transgenic mice according tothe conventional procedure (61).

1.2. PCR Conditions

The PCR amplifications were carried out in a buffer solution containing10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μMof each primer, 2 units of Taq polymerase and 1 μg of genomic DNA astemplate. After 30 cycles (30 sec at 94° C., 30 sec at 550° C., 1 min at72° C.) and then 1 cycle (30 sec at 90° C., 30 sec at 550° C., 5 min at72° C.), the amplification products were separated on a 2.2% agarose gelstained with ethidium bromide.

1.3. Analysis of the Genotype of the Mice.

The transgene Cre-LBD[GR(I747/T)] and the wild-type allele and theexcised allele RXR∝^(ΔAF2(LNL)) were detected by PCR. The detection ofthe transgene Cre-LBD[GR(I747/T)] was carried out with the aid of thenucleotide primers 5′-ATCCGAAAAGAAAACGTTGA-3′ (SEQ ID NO: 14) and5′-ATCCAGGTTACGGATATAGT-3′ (SEQ ID NO: 15), that of the alleles of theRXR∝ gene using the primers 5′-GGTTCTCCGGCCGCTTGGGT-3′ (SEQ ID NO: 16)and 5′-GAAGGCGATGCGCTGCGAAT-3′ (SEQ IN NO: 17). In the same matter, theexcision of the tk-neo marker delimited by loxP sites was followed byPCR with the aid of the primers A (5′-CAAGGAGCCTCCTTTCTCTA-3′) (SEQ IDNO: 18) and B (5′-CCTGCTCTACCTGGTGACTT-3′) (SEQ ID NO 19. These primersamplify a DNA fragment of 156 base pairs from the wild-type allele and afragment of 190 base pairs from the excised allele RXR∝^(ΔAF2(L)).

2. Results

The recombinase activity of Cre-LBD[GR(I747/T)] was also tested in mice.Thus, transgenic mice expressing the fusion protein Cre-LBD[GR(I747/T)]under the control of the promoter of the human cytomegalovirus “majorIE” gene were established. The structure of the transgene is representedin FIG. 14a. It contains the activating/promoter sequences of the humancytomegalovirus “major IE” gene, an intron of the rabbit β-globin gene,coding sequences of Cre-LBD[GR(I747/T)] and the simian virus SV40polyadenylation signal. The site of initiation of transcription isindicated by an arrow. These mice were then crossed with “reporter” miceso as to demonstrate therein the existence of an induced recombinaseactivity. This “reporter” line contains an insertion of the tk-neoselectable marker, delimited by loxP sites, in the intron situatedbetween exons 8 and 9 of the RXRα gene, on one of its two alleles[RXRα^(ΔAF2(LNL))]. After recombination of the tkneo marker, a loxP siteremains in place and constitutes the excised allele RXRα^(ΔAF2 (L)). Thewild-type RXRα allele and the excised allele may be detectedsimultaneously by PCR amplification using an appropriate primer pair Aand B. FIG. 14b schematically represents this PCR strategy by indicatingthe A and B primers as well as certain restriction sites.

The progeny derived from crossing Cre-LBD[GR(I747/T)] mice and“reporter” mice, described above, containing both the transgeneCre-LBD[GR(I747/T)] and the allele RXRα^(ΔAF2 (LNL)) was identified byestablishing their genotype from a tail biopsy. One of these mice wassubjected, at the age of four weeks, to a daily incraperitonealinjection of 0.3 mg of dexamethasone diluted in 100 μl of vegetable oil,for five days. A tail biopsy was taken the day before the firstinjection, as well as the day after the fifth injection. The DNAisolated from these samples was analyzed by PCR with the aid of theoligonucleotides A and B (FIG. 15, lanes 2 and 3) to determine if thetreatment induces the deletion of the tk-neo marker. Lane 1 correspondsto a control reaction, carried out without DNA. The position of the PCRproducts amplified using the wild-type RXRα allele and the excisedallele RXRα^(ΔAF2 (L)) are indicated. The size marker (lane 4)corresponds to the 1 kb ladder (GibcoBRL). The size of the fragments isgiven as base pairs. According to FIG. 15, the induced excision indeedtook place after treatment of the mouse tested with dexamethasone.Indeed, the analysis of the DNA fragments amplified from the tail beforetreatment reveals only the presence of the wild-type RXRα allele whereasthe analysis of the tail of the same mouse, after treatment, indicatesthe presence of the excised allele RXRα^(ΔAF2 (LNL)) (FIG. 15). Thus, itappears that the chimeric recombinase Cre-LBD[GR(I747/T)] exhibits nobasic constitutive activity in the presence of the endogenous ligands.It is, however, induced and active in mice by treatment withdexamethasone.

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19 2334 base pairs nucleic acid single linear cDNA CDS 1..2331 - 1..2331/note= “GR(I747/T) (Human glucocorticoid receptor mutated at position747).” 1 ATG GAC TCC AAA GAA TCA TTA ACT CCT GGT AGA GAA GAA AAC CCC AGC48 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser 1 510 15 AGT GTG CTT GCT CAG GAG AGG GGA GAT GTG ATG GAC TTC TAT AAA ACC 96Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr 20 25 30CTA AGA GGA GGA GCT ACT GTG AAG GTT TCT GCG TCT TCA CCC TCA CTG 144 LeuArg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu 35 40 45 GCTGTC GCT TCT CAA TCA GAC TCC AAG CAG CGA AGA CTT TTG GTT GAT 192 Ala ValAla Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp 50 55 60 TTT CCAAAA GGC TCA GTA AGC AAT GCG CAG CAG CCA GAT CTG TCC AAA 240 Phe Pro LysGly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys 65 70 75 80 GCA GTTTCA CTC TCA ATG GGA CTG TAT ATG GGA GAG ACA GAA ACA AAA 288 Ala Val SerLeu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys 85 90 95 GTG ATG GGAAAT GAC CTG GGA TTC CCA CAG CAG GGC CAA ATC AGC CTT 336 Val Met Gly AsnAsp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu 100 105 110 TCC TCG GGGGAA ACA GAC TTA AAG CTT TTG GAA GAA AGC ATT GCA AAC 384 Ser Ser Gly GluThr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn 115 120 125 CTC AAT AGGTCG ACC AGT GTT CCA GAG AAC CCC AAG AGT TCA GCA TCC 432 Leu Asn Arg SerThr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser 130 135 140 ACT GCT GTGTCT GCT GCC CCC ACA GAG AAG GAG TTT CCA AAA ACT CAC 480 Thr Ala Val SerAla Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 TCT GATGTA TCT TCA GAA CAG CAA CAT TTG AAG GGC CAG ACT GGC ACC 528 Ser Asp ValSer Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr 165 170 175 AAC GGTGGC AAT GTG AAA TTG TAT ACC ACA GAC CAA AGC ACC TTT GAC 576 Asn Gly GlyAsn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp 180 185 190 ATT TTGCAG GAT TTG GAG TTT TCT TCT GGG TCC CCA GGT AAA GAG ACG 624 Ile Leu GlnAsp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr 195 200 205 AAT GAGAGT CCT TGG AGA TCA GAC CTG TTG ATA GAT GAA AAC TGT TTG 672 Asn Glu SerPro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu 210 215 220 CTT TCTCCT CTG GCG GGA GAA GAC GAT TCA TTC CTT TTG GAA GGA AAC 720 Leu Ser ProLeu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 TCGAAT GAG GAC TGC AAG CCT CTC ATT TTA CCG GAC ACT AAA CCC AAA 768 Ser AsnGlu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys 245 250 255 ATTAAG GAT AAT GGA GAT CTG GTT TTG TCA AGC CCC AGT AAT GTA ACA 816 Ile LysAsp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr 260 265 270 CTGCCC CAA GTG AAA ACA GAA AAA GAA GAT TTC ATC GAA CTC TGC ACC 864 Leu ProGln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr 275 280 285 CCTGGG GTA ATT AAG CAA GAG AAA CTG GGC ACA GTT TAC TGT CAG GCA 912 Pro GlyVal Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala 290 295 300 AGCTTT CCT GGA GCA AAT ATA ATT GGT AAT AAA ATG TCT GCC ATT TCT 960 Ser PhePro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320GTT CAT GGT GTG AGT ACC TCT GGA GGA CAG ATG TAC CAC TAT GAC ATG 1008 ValHis Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met 325 330 335AAT ACA GCA TCC CTT TCT CAA CAG CAG GAT CAG AAG CCT ATT TTT AAT 1056 AsnThr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn 340 345 350GTC ATT CCA CCA ATT CCC GTT GGT TCC GAA AAT TGG AAT AGG TGC CAA 1104 ValIle Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln 355 360 365GGA TCT GGA GAT GAC AAC TTG ACT TCT CTG GGG ACT CTG AAC TTC CCT 1152 GlySer Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro 370 375 380GGT CGA ACA GTT TTT TCT AAT GGC TAT TCA AGC CCC AGC ATG AGA CCA 1200 GlyArg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395400 GAT GTA AGC TCT CCT CCA TCC AGC TCC TCA ACA GCA ACA ACA GGA CCA 1248Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro 405 410415 CCT CCC AAA CTC TGC CTG GTG TGC TCT GAT GAA GCT TCA GGA TGT CAT 1296Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His 420 425430 TAT GGA GTC TTA ACT TGT GGA AGC TGT AAA GTT TTC TTC AAA AGA GCA 1344Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala 435 440445 GTG GAA GGA CAG CAC AAT TAC CTA TGT GCT GGA AGG AAT GAT TGC ATC 1392Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile 450 455460 ATC GAT AAA ATT CGA AGA AAA AAC TGC CCA GCA TGC CGC TAT CGA AAA 1440Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470475 480 TGT CTT CAG GCT GGA ATG AAC CTG GAA GCT CGA AAA ACA AAG AAA AAA1488 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys 485490 495 ATA AAA GGA ATT CAG CAG GCC ACT ACA GGA GTC TCA CAA GAA ACC TCT1536 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser 500505 510 GAA AAT CCT GGT AAC AAA ACA ATA GTT CCT GCA ACG TTA CCA CAA CTC1584 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu 515520 525 ACC CCT ACC CTG GTG TCA CTG TTG GAG GTT ATT GAA CCT GAA GTG TTA1632 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu 530535 540 TAT GCA GGA TAT GAT AGC TCT GTT CCA GAC TCA ACT TGG AGG ATC ATG1680 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545550 555 560 ACT ACG CTC AAC ATG TTA GGA GGG CGG CAA GTG ATT GCA GCA GTGAAA 1728 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys565 570 575 TGG GCA AAG GCA ATA CCA GGT TTC AGG AAC TTA CAC CTG GAT GACCAA 1776 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln580 585 590 ATG ACC CTA CTG CAG TAC TCC TGG ATG TTT CTT ATG GCA TTT GCTCTG 1824 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu595 600 605 GGG TGG AGA TCA TAT AGA CAA TCA AGT GCA AAC CTG CTG TGT TTTGCT 1872 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala610 615 620 CCT GAT CTG ATT ATT AAT GAG CAG AGA ATG ACT CTA CCC TGC ATGTAC 1920 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr625 630 635 640 GAC CAA TGT AAA CAC ATG CTG TAT GTT TCC TCT GAG TTA CACAGG CTT 1968 Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His ArgLeu 645 650 655 CAG GTA TCT TAT GAA GAG TAT CTC TGT ATG AAA ACC TTA CTGCTT CTC 2016 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu LeuLeu 660 665 670 TCT TCA GTT CCT AAG GAC GGT CTG AAG AGC CAA GAG CTA TTTGAT GAA 2064 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe AspGlu 675 680 685 ATT AGA ATG ACC TAC ATC AAA GAG CTA GGA AAA GCC ATT GTCAAG AGG 2112 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val LysArg 690 695 700 GAA GGA AAC TCC AGC CAG AAC TGG CAG CGG TTT TAT CAA CTGACA AAA 2160 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu ThrLys 705 710 715 720 CTC TTG GAT TCT ATG CAT GAA GTG GTT GAA AAT CTC CTTAAC TAT TGC 2208 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu AsnTyr Cys 725 730 735 TTC CAA ACA TTT TTG GAT AAG ACC ATG TCC ACC GAG TTCCCC GAG ATG 2256 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Thr Glu Phe ProGlu Met 740 745 750 TTA GCT GAA ATC ATC ACC AAT CAG ATA CCA AAA TAT TCAAAT GGA AAT 2304 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser AsnGly Asn 755 760 765 ATC AAA AAA CTT CTG TTT CAT CAA AAG TGA 2334 Ile LysLys Leu Leu Phe His Gln Lys 770 775 777 amino acids amino acid linearprotein 2 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn ProSer 1 5 10 15 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe TyrLys Thr 20 25 30 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser ProSer Leu 35 40 45 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu LeuVal Asp 50 55 60 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp LeuSer Lys 65 70 75 80 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu ThrGlu Thr Lys 85 90 95 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly GlnIle Ser Leu 100 105 110 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu GluSer Ile Ala Asn 115 120 125 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn ProLys Ser Ser Ala Ser 130 135 140 Thr Ala Val Ser Ala Ala Pro Thr Glu LysGlu Phe Pro Lys Thr His 145 150 155 160 Ser Asp Val Ser Ser Glu Gln GlnHis Leu Lys Gly Gln Thr Gly Thr 165 170 175 Asn Gly Gly Asn Val Lys LeuTyr Thr Thr Asp Gln Ser Thr Phe Asp 180 185 190 Ile Leu Gln Asp Leu GluPhe Ser Ser Gly Ser Pro Gly Lys Glu Thr 195 200 205 Asn Glu Ser Pro TrpArg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu 210 215 220 Leu Ser Pro LeuAla Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 Ser AsnGlu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys 245 250 255 IleLys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr 260 265 270Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr 275 280285 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala 290295 300 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser305 310 315 320 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His TyrAsp Met 325 330 335 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys ProIle Phe Asn 340 345 350 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn TrpAsn Arg Cys Gln 355 360 365 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu GlyThr Leu Asn Phe Pro 370 375 380 Gly Arg Thr Val Phe Ser Asn Gly Tyr SerSer Pro Ser Met Arg Pro 385 390 395 400 Asp Val Ser Ser Pro Pro Ser SerSer Ser Thr Ala Thr Thr Gly Pro 405 410 415 Pro Pro Lys Leu Cys Leu ValCys Ser Asp Glu Ala Ser Gly Cys His 420 425 430 Tyr Gly Val Leu Thr CysGly Ser Cys Lys Val Phe Phe Lys Arg Ala 435 440 445 Val Glu Gly Gln HisAsn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile 450 455 460 Ile Asp Lys IleArg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 Cys LeuGln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys 485 490 495 IleLys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser 500 505 510Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu 515 520525 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu 530535 540 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met545 550 555 560 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala AlaVal Lys 565 570 575 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His LeuAsp Asp Gln 580 585 590 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu MetAla Phe Ala Leu 595 600 605 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala AsnLeu Leu Cys Phe Ala 610 615 620 Pro Asp Leu Ile Ile Asn Glu Gln Arg MetThr Leu Pro Cys Met Tyr 625 630 635 640 Asp Gln Cys Lys His Met Leu TyrVal Ser Ser Glu Leu His Arg Leu 645 650 655 Gln Val Ser Tyr Glu Glu TyrLeu Cys Met Lys Thr Leu Leu Leu Leu 660 665 670 Ser Ser Val Pro Lys AspGly Leu Lys Ser Gln Glu Leu Phe Asp Glu 675 680 685 Ile Arg Met Thr TyrIle Lys Glu Leu Gly Lys Ala Ile Val Lys Arg 690 695 700 Glu Gly Asn SerSer Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 Leu LeuAsp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys 725 730 735 PheGln Thr Phe Leu Asp Lys Thr Met Ser Thr Glu Phe Pro Glu Met 740 745 750Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn 755 760765 Ile Lys Lys Leu Leu Phe His Gln Lys 770 775 2004 base pairs nucleicacid single linear cDNA CDS 1..2001 - 1..2001 /note= “CRE-LBD(GR(I747/T) (Fusion protein for the LBD domain of the humanglucocorticoid receptor mutated at position 747 and the Cre recombinaseprotein).” 3 ATG TCC AAT TTA CTG ACC GTA CAC CAA AAT TTG CCT GCA TTA CCGGTC 48 Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val780 785 790 GAT GCA ACG AGT GAT GAG GTT CGC AAG AAC CTG ATG GAC ATG TTCAGG 96 Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg795 800 805 GAT CGC CAG GCG TTT TCT GAG CAT ACC TGG AAA ATG CTT CTG TCCGTT 144 Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val810 815 820 825 TGC CGG TCG TGG GCG GCA TGG TGC AAG TTG AAT AAC CGG AAATGG TTT 192 Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys TrpPhe 830 835 840 CCC GCA GAA CCT GAA GAT GTT CGC GAT TAT CTT CTA TAT CTTCAG GCG 240 Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu GlnAla 845 850 855 CGC GGT CTG GCA GTA AAA ACT ATC CAG CAA CAT TTG GGC CAGCTA AAC 288 Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln LeuAsn 860 865 870 ATG CTT CAT CGT CGG TCC GGG CTG CCA CGA CCA AGT GAC AGCAAT GCT 336 Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser AsnAla 875 880 885 GTT TCA CTG GTT ATG CGG CGG ATC CGA AAA GAA AAC GTT GATGCC GGT 384 Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp AlaGly 890 895 900 905 GAA CGT GCA AAA CAG GCT CTA GCG TTC GAA CGC ACT GATTTC GAC CAG 432 Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp PheAsp Gln 910 915 920 GTT CGT TCA CTC ATG GAA AAT AGC GAT CGC TGC CAG GATATA CGT AAT 480 Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp IleArg Asn 925 930 935 CTG GCA TTT CTG GGG ATT GCT TAT AAC ACC CTG TTA CGTATA GCC GAA 528 Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg IleAla Glu 940 945 950 ATT GCC AGG ATC AGG GTT AAA GAT ATC TCA CGT ACT GACGGT GGG AGA 576 Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp GlyGly Arg 955 960 965 ATG TTA ATC CAT ATT GGC AGA ACG AAA ACG CTG GTT AGCACC GCA GGT 624 Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser ThrAla Gly 970 975 980 985 GTA GAG AAG GCA CTT AGC CTG GGG GTA ACT AAA CTGGTC GAG CGA TGG 672 Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu ValGlu Arg Trp 990 995 1000 ATT TCC GTC TCT GGT GTA GCT GAT GAT CCG AAT AACTAC CTG TTT TGC 720 Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn TyrLeu Phe Cys 1005 1010 1015 CGG GTC AGA AAA AAT GGT GTT GCC GCG CCA TCTGCC ACC AGC CAG CTA 768 Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser AlaThr Ser Gln Leu 1020 1025 1030 TCA ACT CGC GCC CTG GAA GGG ATT TTT GAAGCA ACT CAT CGA TTG ATT 816 Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu AlaThr His Arg Leu Ile 1035 1040 1045 TAC GGC GCT AAG GAT GAC TCT GGT CAGAGA TAC CTG GCC TGG TCT GGA 864 Tyr Gly Ala Lys Asp Asp Ser Gly Gln ArgTyr Leu Ala Trp Ser Gly 1050 1055 1060 1065 CAC AGT GCC CGT GTC GGA GCCGCG CGA GAT ATG GCC CGC GCT GGA GTT 912 His Ser Ala Arg Val Gly Ala AlaArg Asp Met Ala Arg Ala Gly Val 1070 1075 1080 TCA ATA CCG GAG ATC ATGCAA GCT GGT GGC TGG ACC AAT GTA AAT ATT 960 Ser Ile Pro Glu Ile Met GlnAla Gly Gly Trp Thr Asn Val Asn Ile 1085 1090 1095 GTC ATG AAC TAT ATCCGT AAC CTG GAT AGT GAA ACA GGG GCA ATG GTG 1008 Val Met Asn Tyr Ile ArgAsn Leu Asp Ser Glu Thr Gly Ala Met Val 1100 1105 1110 CGC CTG CTG GAAGAT GGC GAT CTC GAG ATT CAG CAG GCC ACT ACA GGA 1056 Arg Leu Leu Glu AspGly Asp Leu Glu Ile Gln Gln Ala Thr Thr Gly 1115 1120 1125 GTC TCA CAAGAA ACC TCT GAA AAT CCT GGT AAC AAA ACA ATA GTT CCT 1104 Val Ser Gln GluThr Ser Glu Asn Pro Gly Asn Lys Thr Ile Val Pro 1130 1135 1140 1145 GCAACG TTA CCA CAA CTC ACC CCT ACC CTG GTG TCA CTG TTG GAG GTT 1152 Ala ThrLeu Pro Gln Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val 1150 1155 1160ATT GAA CCT GAA GTG TTA TAT GCA GGA TAT GAT AGC TCT GTT CCA GAC 1200 IleGlu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp 1165 11701175 TCA ACT TGG AGG ATC ATG ACT ACG CTC AAC ATG TTA GGA GGG CGG CAA1248 Ser Thr Trp Arg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gln1180 1185 1190 GTG ATT GCA GCA GTG AAA TGG GCA AAG GCA ATA CCA GGT TTCAGG AAC 1296 Val Ile Ala Ala Val Lys Trp Ala Lys Ala Ile Pro Gly Phe ArgAsn 1195 1200 1205 TTA CAC CTG GAT GAC CAA ATG ACC CTA CTG CAG TAC TCCTGG ATG TTT 1344 Leu His Leu Asp Asp Gln Met Thr Leu Leu Gln Tyr Ser TrpMet Phe 1210 1215 1220 1225 CTT ATG GCA TTT GCT CTG GGG TGG AGA TCA TATAGA CAA TCA AGT GCA 1392 Leu Met Ala Phe Ala Leu Gly Trp Arg Ser Tyr ArgGln Ser Ser Ala 1230 1235 1240 AAC CTG CTG TGT TTT GCT CCT GAT CTG ATTATT AAT GAG CAG AGA ATG 1440 Asn Leu Leu Cys Phe Ala Pro Asp Leu Ile IleAsn Glu Gln Arg Met 1245 1250 1255 ACT CTA CCC TGC ATG TAC GAC CAA TGTAAA CAC ATG CTG TAT GTT TCC 1488 Thr Leu Pro Cys Met Tyr Asp Gln Cys LysHis Met Leu Tyr Val Ser 1260 1265 1270 TCT GAG TTA CAC AGG CTT CAG GTATCT TAT GAA GAG TAT CTC TGT ATG 1536 Ser Glu Leu His Arg Leu Gln Val SerTyr Glu Glu Tyr Leu Cys Met 1275 1280 1285 AAA ACC TTA CTG CTT CTC TCTTCA GTT CCT AAG GAC GGT CTG AAG AGC 1584 Lys Thr Leu Leu Leu Leu Ser SerVal Pro Lys Asp Gly Leu Lys Ser 1290 1295 1300 1305 CAA GAG CTA TTT GATGAA ATT AGA ATG ACC TAC ATC AAA GAG CTA GGA 1632 Gln Glu Leu Phe Asp GluIle Arg Met Thr Tyr Ile Lys Glu Leu Gly 1310 1315 1320 AAA GCC ATT GTCAAG AGG GAA GGA AAC TCC AGC CAG AAC TGG CAG CGG 1680 Lys Ala Ile Val LysArg Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg 1325 1330 1335 TTT TAT CAACTG ACA AAA CTC TTG GAT TCT ATG CAT GAA GTG GTT GAA 1728 Phe Tyr Gln LeuThr Lys Leu Leu Asp Ser Met His Glu Val Val Glu 1340 1345 1350 AAT CTCCTT AAC TAT TGC TTC CAA ACA TTT TTG GAT AAG ACC ATG TCC 1776 Asn Leu LeuAsn Tyr Cys Phe Gln Thr Phe Leu Asp Lys Thr Met Ser 1355 1360 1365 ACCGAG TTC CCC GAG ATG TTA GCT GAA ATC ATC ACC AAT CAG ATA CCA 1824 Thr GluPhe Pro Glu Met Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro 1370 1375 13801385 AAA TAT TCA AAT GGA AAT ATC AAA AAA CTT CTG TTT CAT CAA AAG GGT1872 Lys Tyr Ser Asn Gly Asn Ile Lys Lys Leu Leu Phe His Gln Lys Gly1390 1395 1400 ACC AGC CGT GGA GGG GCA TCC GTG GAG GAG ACG GAC CAA AGCCAC TTG 1920 Thr Ser Arg Gly Gly Ala Ser Val Glu Glu Thr Asp Gln Ser HisLeu 1405 1410 1415 GCC ACT GCG GGC TCT ACT TCA TCG CAT TCC TTG CAA AAGTAT TAC ATC 1968 Ala Thr Ala Gly Ser Thr Ser Ser His Ser Leu Gln Lys TyrTyr Ile 1420 1425 1430 ACG GGG GAG GCA GAG GGT TTC CCT GCC ACA GTC TGA2004 Thr Gly Glu Ala Glu Gly Phe Pro Ala Thr Val 1435 1440 667 aminoacids amino acid linear protein 4 Met Ser Asn Leu Leu Thr Val His GlnAsn Leu Pro Ala Leu Pro Va 1 5 10 15 Asp Ala Thr Ser Asp Glu Val Arg LysAsn Leu Met Asp Met Phe Arg 20 25 30 Asp Arg Gln Ala Phe Ser Glu His ThrTrp Lys Met Leu Leu Ser Val 35 40 45 Cys Arg Ser Trp Ala Ala Trp Cys LysLeu Asn Asn Arg Lys Trp Phe 50 55 60 Pro Ala Glu Pro Glu Asp Val Arg AspTyr Leu Leu Tyr Leu Gln Ala 65 70 75 80 Arg Gly Leu Ala Val Lys Thr IleGln Gln His Leu Gly Gln Leu Asn 85 90 95 Met Leu His Arg Arg Ser Gly LeuPro Arg Pro Ser Asp Ser Asn Ala 100 105 110 Val Ser Leu Val Met Arg ArgIle Arg Lys Glu Asn Val Asp Ala Gly 115 120 125 Glu Arg Ala Lys Gln AlaLeu Ala Phe Glu Arg Thr Asp Phe Asp Gln 130 135 140 Val Arg Ser Leu MetGlu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn 145 150 155 160 Leu Ala PheLeu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu 165 170 175 Ile AlaArg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg 180 185 190 MetLeu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly 195 200 205Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp 210 215220 Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys 225230 235 240 Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser GlnLeu 245 250 255 Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His ArgLeu Ile 260 265 270 Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu AlaTrp Ser Gly 275 280 285 His Ser Ala Arg Val Gly Ala Ala Arg Asp Met AlaArg Ala Gly Val 290 295 300 Ser Ile Pro Glu Ile Met Gln Ala Gly Gly TrpThr Asn Val Asn Ile 305 310 315 320 Val Met Asn Tyr Ile Arg Asn Leu AspSer Glu Thr Gly Ala Met Val 325 330 335 Arg Leu Leu Glu Asp Gly Asp LeuGlu Ile Gln Gln Ala Thr Thr Gly 340 345 350 Val Ser Gln Glu Thr Ser GluAsn Pro Gly Asn Lys Thr Ile Val Pro 355 360 365 Ala Thr Leu Pro Gln LeuThr Pro Thr Leu Val Ser Leu Leu Glu Val 370 375 380 Ile Glu Pro Glu ValLeu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp 385 390 395 400 Ser Thr TrpArg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gln 405 410 415 Val IleAla Ala Val Lys Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn 420 425 430 LeuHis Leu Asp Asp Gln Met Thr Leu Leu Gln Tyr Ser Trp Met Phe 435 440 445Leu Met Ala Phe Ala Leu Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala 450 455460 Asn Leu Leu Cys Phe Ala Pro Asp Leu Ile Ile Asn Glu Gln Arg Met 465470 475 480 Thr Leu Pro Cys Met Tyr Asp Gln Cys Lys His Met Leu Tyr ValSer 485 490 495 Ser Glu Leu His Arg Leu Gln Val Ser Tyr Glu Glu Tyr LeuCys Met 500 505 510 Lys Thr Leu Leu Leu Leu Ser Ser Val Pro Lys Asp GlyLeu Lys Ser 515 520 525 Gln Glu Leu Phe Asp Glu Ile Arg Met Thr Tyr IleLys Glu Leu Gly 530 535 540 Lys Ala Ile Val Lys Arg Glu Gly Asn Ser SerGln Asn Trp Gln Arg 545 550 555 560 Phe Tyr Gln Leu Thr Lys Leu Leu AspSer Met His Glu Val Val Glu 565 570 575 Asn Leu Leu Asn Tyr Cys Phe GlnThr Phe Leu Asp Lys Thr Met Ser 580 585 590 Thr Glu Phe Pro Glu Met LeuAla Glu Ile Ile Thr Asn Gln Ile Pro 595 600 605 Lys Tyr Ser Asn Gly AsnIle Lys Lys Leu Leu Phe His Gln Lys Gly 610 615 620 Thr Ser Arg Gly GlyAla Ser Val Glu Glu Thr Asp Gln Ser His Leu 625 630 635 640 Ala Thr AlaGly Ser Thr Ser Ser His Ser Leu Gln Lys Tyr Tyr Ile 645 650 655 Thr GlyGlu Ala Glu Gly Phe Pro Ala Thr Val 660 665 6094 base pairs nucleic acidsingle circular cDNA - 1..6091 /note= “pCRE-LBD[GR(I747/T] (Plasmid forexpressing the fusion protein Cre-LBD(GR(I747/ T)].” 5 GTCGACTTCTGAGGCGGAAA GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG 60 TCCCCAGGCTCCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAAC 120 AGGTGTGGAAAGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAA 180 TAGTCAGCAACCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAG 240 TCCGCCCATTCTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGC 300 GCCTCGGCCTCTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTT 360 TGCAAAAAGCTGGATCGATC CTGAGAACTT CAGGGTGAGT TTGGGGACCC TTGATTGTT 420 TTTCTTTTTCGCTATTGTAA AATTCATGTT ATATGGAGGG GGCAAAGTTT TCAGGGTGT 480 GTTTAGAATGGGAAGATGTC CCTTGTATCA CCATGGACCC TCATGATAAT TTTGTTTCT 540 TCACTTTCTACTCTGTTGAC AACCATTGTC TCCTCTTATT TTCTTTTCAT TTTCTGTAA 600 TTTTTCGTTAAACTTTAGCT TGCATTTGTA ACGAATTTTT AAATTCACTT TTGTTTATT 660 GTCAGATTGTAAGTACTTTC TCTAATCACT TTTTTTTCAA GGCAATCAGG GTATATTAT 720 TTGTACTTCAGCACAGTTTT AGAGAACAAT TGTTATAATT AAATGATAAG GTAGAATAT 780 TCTGCATATAAATTCTGGCT GGCGTGGAAA TATTCTTATT GGTAGAAACA ACTACATCC 840 GGTCATCATCCTGCCTTTCT CTTTATGGTT ACAATGATAT ACACTGTTTG AGATGAGGA 900 AAAATACTCTGAGTCCAAAC CGGGCCCCTC TGCTAACCAT GTTCATGCCT TCTTCTTTT 960 CCTACAGCTCCTGGGCAACG TGCTGGTTAT TGTGCTGTCT CATCATTTTG GCAAAGAA 1020 GTAATACGACTCACTATAGG GCGAATTCCA CCATGTCCAA TTTACTGACC GTACACCA 1080 ATTTGCCTGCATTACCGGTC GATGCAACGA GTGATGAGGT TCGCAAGAAC CTGATGGA 1140 TGTTCAGGGATCGCCAGGCG TTTTCTGAGC ATACCTGGAA AATGCTTCTG TCCGTTTG 1200 GGTCGTGGGCGGCATGGTGC AAGTTGAATA ACCGGAAATG GTTTCCCGCA GAACCTGA 1260 ATGTTCGCGATTATCTTCTA TATCTTCAGG CGCGCGGTCT GGCAGTAAAA ACTATCCA 1320 AACATTTGGGCCAGCTAAAC ATGCTTCATC GTCGGTCCGG GCTGCCACGA CCAAGTGA 1380 GCAATGCTGTTTCACTGGTT ATGCGGCGGA TCCGAAAAGA AAACGTTGAT GCCGGTGA 1440 GTGCAAAACAGGCTCTAGCG TTCGAACGCA CTGATTTCGA CCAGGTTCGT TCACTCAT 1500 AAAATAGCGATCGCTGCCAG GATATACGTA ATCTGGCATT TCTGGGGATT GCTTATAA 1560 CCCTGTTACGTATAGCCGAA ATTGCCAGGA TCAGGGTTAA AGATATCTCA CGTACTGA 1620 GTGGGAGAATGTTAATCCAT ATTGGCAGAA CGAAAACGCT GGTTAGCACC GCAGGTGT 1680 AGAAGGCACTTAGCCTGGGG GTAACTAAAC TGGTCGAGCG ATGGATTTCC GTCTCTGG 1740 TAGCTGATGATCCGAATAAC TACCTGTTTT GCCGGGTCAG AAAAAATGGT GTTGCCGC 1800 CATCTGCCACCAGCCAGCTA TCAACTCGCG CCCTGGAAGG GATTTTTGAA GCAACTCA 1860 GATTGATTTACGGCGCTAAG GATGACTCTG GTCAGAGATA CCTGGCCTGG TCTGGACA 1920 GTGCCCGTGTCGGAGCCGCG CGAGATATGG CCCGCGCTGG AGTTTCAATA CCGGAGAT 1980 TGCAAGCTGGTGGCTGGACC AATGTAAATA TTGTCATGAA CTATATCCGT AACCTGGA 2040 GTGAAACAGGGGCAATGGTG CGCCTGCTGG AAGATGGCGA TCTCGAGATT CAGCAGGC 2100 CTACAGGAGTCTCACAAGAA ACCTCTGAAA ATCCTGGTAA CAAAACAATA GTTCCTGC 2160 CGTTACCACAACTCACCCCT ACCCTGGTGT CACTGTTGGA GGTTATTGAA CCTGAAGT 2220 TATATGCAGGATATGATAGC TCTGTTCCAG ACTCAACTTG CAGGATCATG ACTACGCT 2280 ACATGTTAGGAGGGCGGCAA GTGATTGCAG CAGTGAAATG GGCAAAGGCA ATACCAGG 2340 TCAGGAACTTACACCTGGAT GACCAAATGA CCCTACTGCA GTACTCCTGG ATGTTTCT 2400 TGGCATTTGCTCTGGGGTGG AGATCATATA GACAATCAAG TGCAAACCTG CTGTGTTT 2460 CTCCTGATCTGATTATTAAT GAGCAGAGAA TGACTCTACC CTGCATGTAC GACCAATG 2520 AACACATGCTGTATGTTTCC TCTGAGTTAC ACAGGCTTCA GGTATCTTAT GAAGAGTA 2580 TCTGTATGAAAACCTTACTG CTTCTCTCTT CAGTTCCTAA GGACGGTCTG AAGAGCCA 2640 AGCTATTTGATGAAATTAGA ATGACCTACA TCAAAGAGCT AGGAAAAGCC ATTGTCAA 2700 GGGAAGGAAACTCCAGCCAG AACTGGCAGC GGTTTTATCA ACTGACAAAA CTCTTGGA 2760 CTATGCATGAAGTGGTTGAA AATCTCCTTA ACTATTGCTT CCAAACATTT TTGGATAA 2820 CCATGTCCACCGAGTTCCCC GAGATGTTAG CTGAAATCAT CACCAATCAG ATACCAAA 2880 ATTCAAATGGAAATATCAAA AAACTTCTGT TTCATCAAAA GGGTACCAGC CGTGGAGG 2940 CATCCGTGGAGGAGACGGAC CAAAGCCACT TGGCCACTGC GGGCTCTACT TCATCGCA 3000 CCTTGCAAAAGTATTACATC ACGGGGGAGG CAGAGGGTTT CCCTGCCACA GTCTGAGA 3060 TCCCTGGAATTCGGATCTTA TTAAAGCAGA ACTTGTTTAT TGCAGCTTAT AATGGTTA 3120 AATAAAGCAATAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAG 3180 GTGGTTTGTCCAAACTCATC AATGTATCTT ATCATGTCTG GTCGACTCTA GACTCTTC 3240 CTTCCTCGCTCACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGC 3300 ACTCAAAGGCGGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACAT 3360 CAGCAAAAGGCCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTT 3420 ATAGGCTCCGCCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCG 3480 ACCCGACAGGACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTC 3540 CTGTTCCGACCCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGT 3600 CGCTTTCTCATAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAA 3660 TGGGCTGTGTGCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTA 3720 GTCTTGAGTCCAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAA 3780 GGATTAGCAGAGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAA 3840 ACGGCTACACTAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTT 3900 GAAAAAGAGTTGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTT 3960 TTGTTTGCAAGCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGAT 4020 TTTCTACGGGGTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCAT 4080 GATTATCAAAAAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATC 4140 TCTAAAGTATATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGC 4200 CTATCTCAGCGATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTA 4260 TAACTACGATACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGA 4320 CACGCTCACCGGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCG 4380 GAAGTGGTCCTGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGC 4440 GAGTAAGTAGTTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCAT 4500 TGGTGTCACGCTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAG 4560 GAGTTACATGATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGAT 4620 TTGTCAGAAGTAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAA 4680 CTCTTACTGTCATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAA 4740 CATTCTGAGAATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGA 4800 ATACCGCGCCACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGG 4860 GAAAACTCTCAAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGC 4920 CCAACTGATCTTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGG 4980 GGCAAAATGCCGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACT 5040 TCCTTTTTCAATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACAT 5100 TTGAATGTATTTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGT 5160 CACCTGACGTCTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTAT 5220 CGAGGCCCCTTTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGC 5280 CTCCCGGAGACGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTC 5340 GGCGCGTCAGCGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGC 5400 ATTGTACTGAGAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAA 5460 TACCGCATCAGGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTG 5520 AAATCAGCTCATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAA 5580 AATAGACCGAGATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAA 5640 ACGTGGACTCCAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACG 5700 AACCATCACCCTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAA 5760 CTAAAGGGAGCCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAA 5820 AAGGGAAGAAAGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCT 5880 GCGTAACCACCACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCG CGCCATTC 5940 CATTCAGGCTGCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCG CTATTACG 6000 AGCTGGCGAAAGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA GGGTTTTC 6060 AGTCACGACGTTGTAAAACG ACGGCCAGTG AATT 6094 36 base pairs nucleic acid single linearDNA (genomic) - 1..36 /note= “SG52” 6 CTGGAAGATG GCGATCTCGA GATTCAGCAGGCCACT 36 37 base pairs nucleic acid single linear DNA (genomic) - 1..37/note= “SG53” 7 CTGTTTCATC AAAAGGGTAC CAGCCGTGGA GGGGCAT 37 24 basepairs nucleic acid single linear DNA (genomic) - 1..24 /note= “syntheticoligonucleotide” 8 ATTATTATTA CTCGAGTGAT GATG 24 24 base pairs nucleicacid single linear DNA (genomic) - 1..24 /note= “syntheticoligonucleotide” 9 ATCATCATCA TCCGAGTAAT AATA 24 14 base pairs nucleicacid single linear DNA (genomic) - 1..14 /note= “SB211” 10 GGCAAACACTATGG 14 17 base pairs nucleic acid single linear DNA (genomic) - 1..17/note= “PZ105” 11 TTGCGTACTG TCCTCTT 17 20 base pairs nucleic acidsingle linear DNA (genomic) - 1..20 /note= “synthetic oligonucleotide”12 AAGAAGCCCT TGCAGCCCTC 20 40 base pairs nucleic acid single linear DNA(genomic) - 1..40 /note= “synthetic oligonucleotide” 13 AATTATAACTTCGTATAATG TATGCTATAC GAAGTTATTC 40 20 base pairs nucleic acid singlelinear DNA (genomic) - 1..20 /note= “primer” 14 ATCCGAAAAG AAAACGTTGA 2020 base pairs nucleic acid single linear DNA (genomic) - 1..20 /note=“primer” 15 ATCCAGGTTA CGGATATAGT 20 20 base pairs nucleic acid singlelinear DNA (genomic) - 1..20 /note= “primer” 16 GGTTCTCCGG CCGCTTGGGT 2020 base pairs nucleic acid single linear DNA (genomic) - 1..20 /note=“primer” 17 GAAGGCGATG CGCTGCGAAT 20 20 base pairs nucleic acid singlelinear DNA (genomic) - 1..20 /note= “primer A” 18 CAAGGAGCCT CCTTTCTCTA20 20 base pairs nucleic acid single linear DNA (genomic) - 1..20 /note=“primer B” 19 CCTGCTCTAC CTGGTGACTT 20

What is claimed is:
 1. A polynucleotide comprising a nucleic acidsequence coding for a modified nuclear glucocorticoid receptor, whereinsaid nucleic acid sequence is the cDNA sequence of the human nuclearglucocorticoid receptor as represented in SEQ ID NO: 2, so that thetranscriptional activity of the human nuclear glucocorticoid receptor ismore strongly inducible by a synthetic glucocorticoid ligand than by anatural glucocorticoid.
 2. The polynucleotide according to claim 1,wherein said nucleic acid sequence is the cDNA of the human nuclearglucocorticoid receptor as represented in SEQ ID NO:
 1. 3. Apolynucleotide comprising a nucleic acid sequence coding for a ligandbinding domain (LBD) of a nuclear glucocorticoid receptor, said ligandbinding domain comprising a nucleic acid sequence coding for an aminoacid sequence as represented in SEQ ID NO: 2 from no acid 532 to aminoacid 777, so that the activity of said ligand binding domain is morestrongly inducible by binding to a synthetic glucocorticoid ligand thanby binding to a natural glucocorticoid.
 4. The polynucleotide accordingto claim 3, comprising a nucleic acid sequence coding for the ligandbinding domain (LBD) of the human nuclear glucocorticoid receptor havingthe nucleotide sequence as represented in SEQ ID NO: 1 from nucleotide1594 to nucleotide
 2334. 5. A modified nuclear glucocorticoid receptor,comprising an amino acid sequence as represented in SEQ ID NO: 2, sothat the transcriptional activity of said receptor is more stronglyinducible by a synthetic glucocorticoid ligand than by a naturalglucocorticoid ligand.
 6. A ligand binding domain of a nuclearglucocorticoid receptor wherein said ligand binding domain is of thehuman nuclear glucocorticoid receptor and comprises the amino acidsequence of the ligand binding domain of the human glucocorticoidreceptor as represented in SEQ ID NO: 2 from amino acid 532 to aminoacid 777, so that the activity of said ligand binding domain is morestrongly inducible by a synthetic glucocorticoid ligand tan by a naturalglucocorticoid ligand.
 7. A vector system for conditionally expressing aprotein in host cells, the expression of which is induced in thepresence of a synthetic glucocorticoid ligand, and said vector systemcomprising: a first DNA fragment comprising a nucleic acid sequencecoding for said protein under the control of elements ensuring itsexpression in said host cells, said elements ensuring its expressioncomprising a sequence for control of transcription (RE) being recognizedby a modified glucocorticoid receptor mutated in the region of theligand binding domain between the H11 and H12 helices, so that theactivity of said modified receptor is more strongly inducible by asynthetic glucocorticoid ligand than by a natural glucocorticoid ligand,said modified receptor being complexed with said syntheticglucocorticoid ligand, and a second DNA fragment encoding said modifiedglucocorticoid receptor comprising a polynucleotide coding for: theligand binding domain (LBD) region of the human glucocorticoid receptorcomprising an amino acid sequence as represented in SEQ ID NO: 2 fromamino acid 532 to amino acid 777, and the DNA binding domain (DBD)region of the human glucocorticoid receptor comprising an amino acidsequence as represented in SEQ ID NO: 2 from amino acid 421 to aminoacid 487, said polynucleotide being placed under the control of theelements assuring its expression in said host cells, said first andsecond DNA fragments being carried by the same vector or two vectorsseparately.
 8. A method for expressing a foreign protein in human oranimal cells in vitro comprising culturing cells which contain a vectorsystem according to claim 7, in the presence of a syntheticglucocorticoid ligand to induce expression of the foreign protein and,recovering the expressed foreign protein.
 9. The method according toclaim 8, wherein said synthetic ligand is selected from the groupconsisting of dexamethasone, triamcinolone acetonide, RU2836,bimetrazole, deacylcortivazol and fluocinolone acetonide.
 10. Apolynucleotide sequence coding for a polypeptide comprising: (a) anucleic acid encoding a polypeptide wherein said encoded polypeptidecomprises the human nuclear glucocorticoid receptor comprising aminoacid 532 to amino acid 777 of SEQ ID NO:2, wherein the activity of aligand binding domain of the encoded polypeptide is more stronglyinducible by binding to a synthetic glucocorticoid ligand than bybinding to a natural glucocorticoid; and (b) a second nucleic acidcomprising a DNA binding domain.
 11. The polynucleotide according toclaim 10, wherein said DNA binding domain is of a site-specificDNA-binding factor.
 12. The polynucleotide according to claim 11,wherein said site-specific DNA-binding factor is the yeast Gal 4protein.
 13. A polypeptide comprising (a) a ligand binding domain (LBD)of a nuclear glucocorticoid receptor, said ligand binding domaincomprising the amino acid sequence of the human nuclear glucocorticoidreceptor as set forth in SEQ ID NO: 2 from amino acid 532 to amino acid777, so that the activity of said ligand binding domain is more stronglyinducible by binding to a synthetic glucocorticoid ligand than bybinding to a natural glucocorticoid and (b) a DNA binding domain. 14.The polypeptide according to claim 13, wherein said DNA binding domainis of a nuclear glucocorticoid receptor.
 15. The polypeptide accordingto claim 13, wherein said DNA binding domain is of a site-specificDNA-binding factor.
 16. The polypeptide according to claim 15, whereinsaid site-specific DNA-binding factor is the yeast Gal 4 protein.
 17. Avector system for conditionally expressing a protein in host cells, saidvector system being inducible in the presence of a syntheticglucocorticoid ligand, said vector system comprising: a first DNAfragment comprising a nucleic acid sequence coding for said proteinunder the control of elements ensuring the expression in said hostcells, said elements ensuring its expression comprising a sequence forcontrol of transcription (RE) being recognized by the DNA binding domainof a polypeptide comprising (a) the ligand binding domain (LBD) regionof the human glucocorticoid receptor comprising an amino acid sequenceas represented in SEQ ID NO: 2 from amino acid 532 to amino acid 777, sothat the activity of said ligand binding domain is more stronglyinducible by binding to a synthetic glucocorticoid ligand than bybinding to a natural glucocorticoid and (b) a DNA binding domain of asite-specific DNA-binding factor which binds to said sequence forcontrol of the transcription (RE), said polypeptide being complexed withsaid synthetic glucocorticoid ligand, and a second DNA fragmentcomprising a polynucleotide coding for said polypeptide placed under thecontrol of the elements assuring its expression in said host cells, saidfirst and second DNA fragments being carried by the same vector or twovectors separately.
 18. The vector system according to claim 17 whereinsaid site-specific DNA-binding factor is the yeast protein Gal 4 andsaid sequence for control of transcription (RE) is the 17 m sequencerecognized by the Gal4 transactivator.
 19. The method according to claim8, wherein said vector system is inducible in the presence of asynthetic glucocorticoid ligand, said vector system comprising: a firstDNA fragment comprising a nucleic acid sequence coding for said proteinunder the control of elements ensuring the expression in said hostcells, said elements ensuring its expression comprising a sequence forcontrol of transcription (RE) being recognized by the DNA binding domainof a polypeptide comprising (a) a ligand binding domain (LBD) of anuclear glucocorticoid receptor, said ligand binding domain comprisingan amino acid sequence as represented in SEQ ID NO: 2 from amino acid532 to amino acid 777, so that the activity of said ligand bindingdomain is more strongly inducible by binding to a syntheticglucocorticoid ligand than by binding to a natural glucocorticoid and(b) a DNA binding domain of a site-specific DNA-binding factor whichbinds to said sequence for control of the transition (RE), saidpolypeptide being complexed with said synthetic glucocorticoid ligand,and a second DNA fragment comprising a polynucleotide coding for saidpolypeptide placed under the control of the elements assuring itsexpression in said host cells, said first and second DNA fragments beingcarried by the same vector or two vectors separately; and wherein saidsite-specific DNA-binding factor is the yeast protein Gal 4 and saidsequence for control of transcription (RE) is the 17 m sequencerecognized by the Gal4 transactivator.